Synthesis of four-color 3&#39;-o-allyl modified photocleavable fluorescent nucleotides and related methods

ABSTRACT

This invention provides a process for making 3′-O-allyl-dGTP-PC-Biodopy-FL-510, 3′-O-allyl-dATP-PC-ROX, 3′-O-allyl-dCTP-PC-Bodipy-650 and 3′-O-allyl-dUTP-PC-R6G, and related methods.

This application is a continuation of U.S. Ser. No. 13/186,353, filedJul. 19, 2011, now allowed, which is a continuation of U.S. Ser. No.12/084,338, filed Oct. 16, 2009, now U.S. Pat. No. 7,982,029, issuedJul. 19, 2011, which is a §371 national stage of PCT InternationalApplication No. PCT/US2006/042698, filed Oct. 31, 2006, which claims thebenefit of U.S. Provisional Application No. 60/732,373, filed Oct. 31,2005, the contents of each of which are hereby incorporated by referencein their entirety into this application.

Throughout this application, various publications are referenced inparentheses by number. Full citations for these references may be foundat the end of each experimental section. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application to more fully describe the state of the art towhich this invention pertains.

This invention was made with Government support under Center ofExcellence in Genomic Science Grant No. IP50 HG002806-01 awarded by theNational institutes of Health, U.S. Department of Health and HumanServices. Accordingly, the U.S. Government has certain rights in thisinvention.

REFERENCE TO SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“160111_75416-AAA-PCT-US_Substitute_Sequence_Listing_AC.txt,” which is4.08 kilobytes in size, and which was created Jan. 11, 2016 in theIBM-PC machine format, having an operating system compatibility withMS-Windows, which is contained in the text file filed Jan. 11, 2016 aspart of this application.

BACKGROUND OF THE INVENTION

DNA sequencing is a fundamental tool for biological research and medicaldiagnostics, driving disease gene discovery and gene function studies.DNA sequencing by synthesis (SBS) using reversible fluorescentnucleotide terminators1 is a potentially efficient approach to addressthe limitations of current DNA sequencing techniques, such as throughputand data accuracy. A 3′-O-allyl photocleavable (PC) fluorescentnucleotide analogue, 3′-O-allyl-dUTP-PC-Bodipy-FL-510, as a reversibleterminator for SBS has previously been reported (2). The nucleotide canbe efficiently incorporated by DNA polymerase into a growing DNA strandto terminate the polymerase reaction. After that the fluorophore can bephotocleaved quantitatively by irradiation at 355 nm, and the allylgroup is rapidly and efficiently removed by using a Pd-catalyzedreaction in water to regenerate a free 3′-OH group to reinitiate thepolymerase reaction.

SUMMARY

This invention provides a method for making3′O-allyl-dGTP-PC-Bodipy-FL-510 comprising performing the steps setforth in FIG. 7. This invention also provides a method for making3′-O-allyl-dATP-PC-ROX comprising performing the steps set forth in FIG.8. This invention also provides a method for making3′-O-allyl-dCTP-PC-Bodipy-650 comprising performing the steps set forthin FIG. 9. This invention also provides a method for making3′-O-allyl-dUTP-PC-R6G comprising performing the steps set forth in FIG.10.

This invention also provides a method for making method for determiningthe sequence of a DNA comprising performing the following steps for eachresidue of the DNA to be sequenced:

-   -   (a) contacting the DNA with a DNA polymerase in the presence        of (i) a primer and (ii) four fluorescent nucleotide analogues        under conditions permitting the DNA polymerase to catalyze DNA        synthesis, wherein (1) the nucleotide analogues consist of an        analogue of dGTP, an analogue of dCTP, an analogue of dTTP or        dUTP, and an analogue of dATP, (2) each nucleotide analogue        comprises (i) a base selected from the group consisting of        adenine, guanine, cytosine, thymine or uracil, and analogues        thereof, (ii) a deoxyribose, (iii) a fluorophore photocleavably        attached to the base, and (iv) an allyl moiety bound to the        3′-oxygen of the deoxyribose, so that a nucleotide analogue        complementary to the residue being sequenced is bound to the DNA        by the DNA polymerase, and (3) each of the four analogues has a        predetermined fluorescence wavelength which is different than        the fluorescence wavelengths of the other three analogues;    -   (b) removing unbound nucleotide analogues;    -   (c) determining the identity of the bound nucleotide analogues;        and    -   (d) following step (c), except with respect to the final DNA        residue to be sequenced, (i) chemically cleaving from the bound        nucleotide analogue the allyl moiety bound to the 3′-oxygen atom        of the deoxyribose and (ii) photocleaving the fluorophore from        the bound nucleotide analogue, wherein steps (d) (i) and    -   (d) (ii) can be performed concurrently or in any order, and        step (d) (i) is performed using a Pd catalyst at a pH of about        8.8, thereby determining the sequence of the DNA.

This invention also provides a method for removing an allyl moiety fromthe 3′-oxygen of a nucleotide analogue's deoxyribose moiety comprisingthe step of contacting the nucleotide analogue with a Pd catalyst at apH of about 8.8.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Synthesis of a 3′-O-allyl modified 19-mer oligonucleotide.

FIG. 2: Synthesis of 3′-O-allyl-dUTP-PC-Bodipy-FL-510.

FIG. 3: Schematic representation (left) and step-by-step MALDI-TOF MSresults (right) for the deallylation of a 3′-O-allyl-modifiedoligonucleotide and the use of the deallylated oligonucleotide as aprimer in a polymerase extension reaction. (A) Peak at m/z 5871corresponding to the HPLC-purified 3′-O-allyl modified 19-meroligonucleotide. (B) Peak at m/z 5831 corresponding to the aboveoligonucleotide without the allyl group, obtained after 30 sec ofincubation with Na₂PdCl₄ and TPPTS [P(PhSO₃Na)₃] at 70° C. (C) Peak atm/z 6535 corresponding to the extension of the deallylatedoligonucleotide by Biotin-11-ddGTP using Thermo Sequenase DNApolymerase.

FIG. 4: One entire polymerase reaction cycle using3′-O-allyl-dUTP-PC-Bodipy-FL-510 as a reversible terminator.

FIG. 5: MALDI-TOF MS results for each step of a polymerase reactioncycle using 3′-O-allyl-dUTP-PC-Bodipy-FL-510 as a reversible terminator.(A) Peak at m/z 6787 corresponding to the primer extension product 11obtained using 3′-O-allyl-dUTP-PC-Bodipy-FL-510 and the 90N Polymerase(exo-) A485L/Y409V. (B) Peak at m/z 6292 corresponding to thephotocleavage product 12. (C) Peak at m/z 6252 corresponding to thephotocleavage product without the allyl group 13 obtained after 90 secsof incubation with the catalyst and ligand at 70° C. (D) Peak at m/z7133 corresponding to the extension product 14 from the purifieddeallylated product using dGTP-PC-Bodipy-FL-510 and Thermo Sequenase DNAPolymerase. (E) Peak at m/z 6637 corresponding to the photocleavageproduct 15.

FIG. 6: Structures of four-color 3′-O-allyl modified photocleavablefluorescent nucleotides.

FIG. 7: Synthesis of 3′-O-allyl-dGTP-PC-Bodipy-FL-510 10.

FIG. 8. Synthesis of 3′-O-allyl-dATP-PC-ROX 19.

FIG. 9: Synthesis of 3′-O-allyl-dCTP-PC-Bodipy-650 26.

FIG. 10. Synthesis of 3′-O-allyl-dUTP-PC-R6G 33.

FIG. 11. Polymerase DNA extension reaction using3′-O-allyl-dGTP-PC-Bodipy-FL-510 10 as a reversible terminator.

FIG. 12. A continuous polymerase extension using 10 as a reversibleterminator (left) and MALDI-TOF MS spectra of consecutive extensionphotocleavage and deallylation products (right).

FIG. 13. (Left) Scheme showing 3′-O-allyl-dATP-PC-ROX 19 as a basespecific reversible terminator for DNA primer extension, photocleavageand deallylation; (right) MALDI-TOF MS spectra for incorporation,photocleavage and deallylation products.

FIG. 14. (Left) Scheme showing 3′-O-allyl-dCTP-PC-Bodipy-650 26 as abase specific reversible terminator for DNA primer extension,photocleavage and deallylation; (right) MALDI-TOF MS spectra forincorporation, photocleavage and deallylation products.

FIG. 15. (Left) Scheme showing 3′-O-allyl-dUTP-PC-R6G 33 as a basespecific reversible terminator for DNA primer extension, photocleavageand deallylation; (right) MALDI-TOF MS spectra for incorporation,photocleavage and deallylation products.

FIG. 16: Structures of dGTP-PC-Bodipy-FL-510 (λ_(abs (max)) 502 nm;λ_(em (max))=510 nm), dUTP-PC-R6G (λ_(abs (max))=525 nm;λ_(em (max))=550 nm), dATP-PC-ROX (λ_(abs (max))=575 nm;λ_(em (max))=602 nm), and dCTP-PC-Bodipy-650 (λ_(abs (max))=630 nm;λ_(em (max))=650 nm).

FIG. 17: Synthesis of photocleavable fluorescent nucleotides. (a)acetonitrile or DMF/1 M NaHCO₃ solution; (b) N,N′-disuccinimidylcarbonate (DSC), triethylamine; (c) 0.1 M Na₂CO₃/NaHCO₃ aqueous buffer(pH 8.5-8.7).

FIG. 18: DNA extension reaction performed in solution phase tocharacterize the 4 different photocleavable fluorescent nucleotideanalogues (dUTP-PC-R6G, dGTP-PC-Bodipy-FL-510, dATP-PC-ROX,dCTP-PC-Bodipy-650). After each extension reaction, the DNA extensionproduct is purified by HPLC for MALDI-TOF MS measurement, to verify thatit is the correct extension product. Photolysis is performed to producea DNA product that is used as a primer for the next DNA extensionreaction.

FIG. 19: The polymerase extension scheme (left) and MALDI-TOF MS spectraof the four consecutive extension products and their photocleavageproducts (right). Primer extended with dUTP-PC-R6G (1), and itsphotocleavage product 2; Product 2 extended with dGTP-PC-Bodipy-FL-510(3), and its photocleavage product 4; Product 4 extended withdATP-PC-ROX (5), and its photocleavage product 6; Product 6 extendedwith dCTP-PC-Bodipy-650 (7), and its photocleavage product 8. After 10seconds of irradiation with a laser at 355 nm, photocleavage is completewith all the fluorophores cleaved from the extended DNA products.

FIG. 20: Immobilization of an azido-labeled PCR product on analkynyl-functionalized surface and a ligation reaction between theimmobilized single-stranded DNA template and a loop primer to form aself-priming DNA moiety on the chip. The sequence of the loop primer isshown in (A).

FIG. 21: Schematic representation of SBS on a chip using four PCfluorescent nucleotides (Upper panel) and the scanned fluorescenceimages for each step of SBS on a chip (Lower panel). (1) Incorporationof dATP-PC-ROX; (2) Photocleavage of PC-ROX; (3) Incorporation ofdGTP-PC-Bodipy-FL-510; (4) Photocleavage of PC-Bodipy-FL-510; (5)Incorporation of dATP-PC-ROX; (6) Photocleavage of PC-ROX; (7)Incorporation of dCTP-PC-Bodipy-650; (8) Photocleavage of PC-Bodipy-650;(9) Incorporation of dUTP-PC-R6G; (10) Photocleavage of PC-R6G; (11)Incorporation of dATP-PC-ROX; (12) Photocleavage of PC-ROX; (13)Incorporation of dUTP-PC-R6G; (14) Photocleavage of PC-R6G; (15)Incorporation of dATP-PC-ROX; (16) Photocleavage of PC-ROX; (17)Incorporation of dGTP-PC-Bodipy-FL-510; (18) Photocleavage ofPC-Bodipy-FL-510; (19) Incorporation of dUTP-PC-R6G; (20) Photocleavageof PC-R6G; (21) Incorporation of dCTP-PC-Bodipy-650; (22) Photocleavageof PC-Bodipy-650; (23) Incorporation of dATP-PC-ROX; (24) Photocleavageof PC-ROX.

FIG. 22: 4-Color DNA sequencing data using3′-O-allyl-dGTP-PC-Bodipy-FL-510, 3′-O-allyl-dATP-PC-ROX,3′-O-allyl-dUTP-PC-R6G and 3′-O-allyl-dCTP-PC-Bodipy-650 as reversibleterminators and a 4-color Laser Scanner. Scanned fluorescence images foreach step of SBS on a DNA chip to sequence a DNA template withhomopolymeric regions.

DETAILED DESCRIPTION OF THE INVENTION Terms

The following definitions are presented as an aid in understanding thisinvention:

-   A—Adenine;-   C—Cytosine;-   DNA—Deoxyribonucleic acid;-   G—Guanine;-   PC—Photocleavable-   RNA—Ribonucleic acid;-   SBS—Sequencing by synthesis;-   T—Thymine; and-   U—Uracil.

“Nucleic acid” shall mean any nucleic acid, including, withoutlimitation, DNA, RNA and hybrids thereof. The nucleic acid bases thatform nucleic acid molecules can be the bases A, C, G, T and U, as wellas derivatives thereof. Derivatives of these bases are well known in theart, and are exemplified in PCR Systems, Reagents and Consumables(Perkin Elmer Catalogue 1996 1997, Roche Molecular Systems, Inc.,Branchburg, N.J., USA).

As used herein, “self-priming moiety” shall mean a nucleic acid moietycovalently bound to a nucleic acid to be transcribed, wherein the boundnucleic acid moiety, through its proximity with the transcriptioninitiation site of the nucleic acid to be transcribed, permitstranscription of the nucleic acid under nucleic acidpolymerization-permitting conditions (e.g. the presence of a suitablepolymerase, nucleotides and other reagents). That is, the self-primingmoiety permits the same result (i.e. transcription) as does a non-boundprimer. In one embodiment, the self-priming moiety is a single strandednucleic acid having a hairpin structure. Examples of such self-primingmoieties are shown in the Figures.

“Hybridize” shall mean the annealing of one single-stranded nucleic acidto another nucleic acid based on sequence complementarity. Thepropensity for hybridization between nucleic acids depends on thetemperature and ionic strength of their milieu, the length of thenucleic acids and the degree of complementarity. The effect of theseparameters on hybridization is well known in the art (see Sambrook J,Fritsch E F, Maniatis T. 1989. Molecular cloning: a laboratory manual.Cold Spring Harbor Laboratory Press, New York.)

As used herein, “nucleotide analogue” shall mean an analogue of A, G, C,T or U (that is, an analogue of a nucleotide comprising the base A, G,C, T or U) which is recognized by DNA or RNA polymerase (whichever isapplicable) and incorporated into a strand of DNA or RNA (whichever isappropriate). Examples of nucleotide analogues include, withoutlimitation 7-deaza-adenine, 7-deaza-guanine, the analogues ofdeoxynucleotides shown in FIG. 6, analogues in which a label is attachedthrough a cleavable linker to the 5-position of cytosine or thymine orto the 7-position of deaza-adenine or deaza-guanine, analogues in whicha small chemical moiety such as —CH₂CH═CH₂ is used to cap the —OH groupat the 3′-position of deoxyribose, and analogues of relateddideoxynucleotides. Nucleotide analogues, including dideoxynucleotideanalogues, and DNA polymerase-based DNA sequencing are also described inU.S. Pat. No. 6,664,079.

1,3 dipolar azide-alkyne cycloaddition chemistry is described in WO2005/084367 and PCT/US03/39354, the contents of each of which are herebyincorporated by reference.

All embodiments of U.S. Pat. No. 6,664,079 (the contents of which arehereby incorporated by reference) with regard to sequencing a nucleicacid are specifically envisioned here.

With regard to the synthesis of the nucleotide analogues disclosedherein, other fluorophores or chromophores to be photocleavably attachedto the base of the analogue are envisioned. In addition, combinatorialfluorescence energy tags as described in U.S. Pat. No. 6,627,748 (thecontents of which are hereby incorporated by reference) may be used inplace of the fluorophores described herein.

EMBODIMENTS OF THE INVENTION

This invention provides a method for making3′O-allyl-dGTP-PC-Bodipy-FL-510 comprising performing the steps setforth in FIG. 7. This invention also provides a method for making3′-O-allyl-dATP-PC-ROX comprising performing the steps set forth in FIG.8. This invention also provides a method for making3′-O-allyl-dCTP-PC-Bodipy-650 comprising performing the steps set forthin FIG. 9. This invention also provides a method for making3′-O-allyl-dUTP-PC-R6G comprising performing the steps set forth in FIG.10.

This invention also provides a method for making method for determiningthe sequence of a DNA comprising performing the following steps for eachresidue of the DNA to be sequenced:

-   -   (a) contacting the DNA with a DNA polymerase in the presence        of (i) a primer and (ii) four fluorescent nucleotide analogues        under conditions permitting the DNA polymerase to catalyze DNA        synthesis, wherein (1) the nucleotide analogues consist of an        analogue of dGTP, an analogue of dCTP, an analogue of dTTP or        dUTP, and an analogue of dATP, (2) each nucleotide analogue        comprises (i) a base selected from the group consisting of        adenine, guanine, cytosine, thymine or uracil, and analogues        thereof, (ii) a deoxyribose, (iii) a fluorophore photocleavably        attached to the base, and (iv) an allyl moiety bound to the        3′-oxygen of the deoxyribose, so that a nucleotide analogue        complementary to the residue being sequenced is bound to the DNA        by the DNA polymerase, and (3) each of the four analogues has a        predetermined fluorescence wavelength which is different than        the fluorescence wavelengths of the other three analogues;    -   (b) removing unbound nucleotide analogues;    -   (c) determining the identity of the bound nucleotide analogues;        and    -   (d) following step (c), except with respect to the final DNA        residue to be sequenced, (i) chemically cleaving from the bound        nucleotide analogue the allyl moiety bound to the 3′-oxygen atom        of the deoxyribose and (ii) photocleaving the fluorophore from        the bound nucleotide analogue, wherein steps (d) (i) and    -   (d) (ii) can be performed concurrently or in any order, and        step (d) (i) is performed using a Pd catalyst at a pH of about        8.8, thereby determining the sequence of the DNA.

In one embodiment of the instant method, chemically cleaving the allylmoiety bound to the 3′-oxygen atom is performed using Na₂PdCl₄.

In one embodiment of the instant method, the primer is a self-primingmoiety.

In one embodiment of the instant method, the DNA is bound to a solidsubstrate. In one embodiment of the instant method, the DNA is bound tothe solid substrate via 1,3-dipolar azide-alkyne cycloadditionchemistry. In one embodiment of the instant method, about 1000 or fewercopies of the DNA are bound to the solid substrate.

In one embodiment of the instant method, the four fluorescent nucleotideanalogues are 3′-O-allyl-dGTP-PC-Bodipy-FL-510, 3′-O-allyl-dATP-PC-ROX,3′-O-allyl-dCTP-PC-Bodipy-650 and 3′-O-allyl-dUTP-PC-R6G.

In one embodiment of the instant method, the DNA polymerase is a 9°Npolymerase.

This invention also provides a method for removing an allyl moiety fromthe 3′-oxygen of a nucleotide analogue's deoxyribose moiety comprisingthe step of contacting the nucleotide analogue with a Pd catalyst at apH of about 8.8. In one embodiment of the instant method, the Pdcatalyst is Na₂PdCl₄.

In embodiments of this invention the sequencing methods described can beapplied, mutatis mutandis, to sequencing an RNA molecule or an RNA/DNAhybrid molecule.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS

The design and synthesis of a complete set of four-color 3′-O-allylmodified photocleavable fluorescent nucleotides as reversibleterminators for SBS is disclosed herein.

Example 1 Synopsis

DNA sequencing by synthesis (SBS) offers a new approach for potentialhigh-throughput sequencing applications. In this method, the ability ofan incoming nucleotide to act as a reversible terminator for a DNApolymerase reaction is an important requirement to unambiguouslydetermine the identity of the incorporated nucleotide before the nextnucleotide is added. A free 3′-OH group on the terminal nucleotide ofthe primer is necessary for the DNA polymerase to incorporate anincoming nucleotide. Therefore, if the 3′-OH group of an incomingnucleotide is capped by a chemical moiety, it will cause the polymerasereaction to terminate after the nucleotide is incorporated into the DNAstrand. If the capping group is subsequently removed to generate a free3′-OH, the polymerase reaction will reinitialize. Here, the design andsynthesis of a 3′-modified photocleavable fluorescent nucleotide,3′-O-allyl-dUTP-PC-Bodipy-FL-510, as a reversible terminator for SBS isdisclosed. This nucleotide analogue contains an allyl moiety capping the3′-OH group and a fluorophore Bodipy-FL-510 linked to the 5 position ofthe uracil through a photocleavable 2-nitrobenzyl linker. In addition,it is shown that this nucleotide is a good substrate for a DNApolymerase. After the nucleotide was successfully incorporated into agrowing DNA strand and the fluorophore photocleaved, the allyl group wasremoved using a Pd catalyzed reaction to reinitiate the polymerasereaction, thereby establishing the feasibility of using such nucleotideanalogues as reversible terminators for SBS.

Introduction

The completion of the Human Genome Project (1, 2) has led to anincreased demand for high-throughput and rapid DNA sequencing methods toidentify genetic variants for applications in pharmacogenomics (3),disease gene discovery (4, 5) and gene function studies (6). Currentstate-of-the-art DNA sequencing technologies (7-11) to some extentaddress the accuracy and throughput requirements but suffer limitationswith respect to cost and data quality. Thus, new DNA sequencing approachis required to broaden the applications of genomic information inmedical research and health care. In this regard, DNA sequencing bysynthesis (SBS) offers an alternative approach to possibly address thelimitations of current DNA sequencing techniques. The design of aparallel chip based SBS system, which uses a self-priming DNA templatecovalently linked to the glass surface of a chip and four modifiednucleotides has previously been described (12-14). The nucleotides aremodified such that they have a photocleavable fluorescent moietyattached to the base (5 position of pyrimidines, 7 position of purines)and a chemically cleavable group to cap the 3′-OH. When the correctnucleotide is incorporated in a DNA polymerase reaction, specific to thetemplate sequence, the reaction is temporarily terminated because of thelack of a free 3′-OH group. After the fluorescent signal is detected andthe nucleotide identified, the 3′-OH needs to be regenerated in order tocontinue incorporating the next nucleotide. In Example 3 hereinbelow, itis demonstrated that 4 photocleavable fluorescent nucleotides can beefficiently incorporated by DNA polymerase into a growing DNA strandbase specifically in a polymerase extension reaction, and thefluorophores can be completely removed by photocleavage under near UVirradiation (λ˜355 nm) with high efficiency (15). Using this system in afour-color sequencing assay, accurate identification of multiple basesin a self-priming DNA template covalently attached to a glass surfacecan be achieved.

Another important requirement for this approach to sequence DNAunambiguously is a suitable chemical moiety to cap the 3′-OH of thenucleotide such that it terminates the polymerase reaction to allow theidentification of the incorporated nucleotide. The capping group thenneeds to be efficiently removed to regenerate the 3′-OH thereby allowingthe polymerase reaction to continue. Thus, the photocleavablefluorescent nucleotides used in SBS must be reversible terminators ofthe DNA polymerase reaction to allow the detection of the fluorescentsignal such that the complementary DNA synthesis and sequenceidentification can be efficiently performed in tandem. The principalchallenge posed by this requirement is the incorporation ability of the3′-modified nucleotide by DNA polymerase into the growing DNA strand.The 3′-position on the sugar ring of a nucleotide is very close to theamino acid residues in the active site of the DNA polymerase. This issupported by the 3-D structure of the previously determined ternarycomplexes of rat DNA polymerase, a DNA template-primer, anddideoxycytidine triphosphate (16). Thus, any bulky modification at thisposition provides steric hindrance to the DNA polymerase and preventsthe nucleotide from being incorporated. A second challenge is theefficient removal of the capping group once the fluorescence signal isdetected. Thus, it is important to use a functional group small enoughto present no hindrance to DNA polymerase, stable enough to withstandDNA extension reaction conditions, and able to be removed easily andrapidly to regenerate a free 3′-OH under specific conditions.

Results

Numerous studies have previously been undertaken to identify a3′-modified nucleotide as a substrate for DNA polymerase.3′-O-methyl-nucleotides have been shown to be good substrates forseveral polymerases (17). However, the procedure to chemically cleavethe methyl group is stringent and requires anhydrous conditions. Thus,it is not practical to use a methyl group to cap the 3′-OH group forSBS. It has been reported that nucleotides with ether linkages at the 3′position can be incorporated by some DNA polymerases, while those withester linkages are not generally accepted by most of the polymerasestested (18). Significant efforts have been dedicated to evaluate a widevariety of 3′-modified nucleotides to be used as terminators for variousDNA polymerases and reverse transcriptases but none of the functionalgroups tested have had established methods to regenerate a free 3′-OH(19-22).

It is known that stable chemical functionalities such as allyl(—CH₂—CH═CH₂) and methoxymethyl (—CH₂—O—CH₃) groups can be used to capan OH group, and can be cleaved chemically with high yield (23, 24). Useof such groups as reversible caps for the 3′-OH of the nucleotide forSBS (12) is investigated here, and the establishment of the allyl groupas a 3′-OH capping moiety for the nucleotide analogues that can be usedin SBS is revealed. The choice of this group was based on the fact thatthe allyl moiety, being relatively small, would not provide significanthindrance for the polymerase reaction, and therefore allow the incoming3′-O-allyl modified nucleotide analogue to be accepted by DNApolymerase. Furthermore, it was proposed to remove this group usingcatalytic deallylation. Here, the synthesis of a photocleavablefluorescent nucleotide analogue, 3′-O-allyl-dUTP-PC-Bodipy-FL-510, thatcan be efficiently incorporated by DNA polymerase into a growing DNAstrand is shown. The allyl group can be rapidly and completely removedby a Pd catalyzed reaction to regenerate a 3′-OH group and thedeallylated DNA can then allow reinitiation of the polymerase reactionto incorporate the subsequent nucleotide analogue.

Materials and Methods

All chemicals were purchased from Sigma-Aldrich unless otherwiseindicated. Oligonucleotides used as primers or templates weresynthesized on an EXPEDITE Nucleic Acid Synthesizer (AppliedBiosystems). ¹H NMR spectra were recorded on a Bruker 400 spectrometer,while ¹³C and ³¹P NMR spectra were recorded on a Bruker 300spectrometer. High-resolution MS (HRMS) data were obtained by using aJEOL JMS HX 110A mass spectrometer. Mass measurement of DNA was made ona Voyager DE matrix-assisted laser desorption ionization time-of-flight(MALDI-TOF) mass spectrometer (Applied Biosystems). Photolysis wasperformed using a Spectra Physics GCR-150-30 Nd-YAG laser that generateslight pulses at 355 nm (ca. 50 mJ/pulse, pulse length ca. 7 ns) at afrequency of 30 Hz with a light intensity at ca. 1.5 W/cm². ThermoSequenase DNA Polymerase, HIV-1 and RAV2 Reverse Transcriptases wereobtained from Amersham Biosciences. Therminator, Vent (exo-), Deep Vent(exo-), Bst and Klenow (exo-) fragment DNA Polymerases were obtainedfrom New England Biolabs. 9°N Polymerase (exo-) A485L/Y409V wasgenerously provided by New England Biolabs. Sequenase V2 DNA Polymerase,M-MulV and AMV Reverse Transcriptases were obtained from USB Corporation(Cleveland, Ohio). Tfl and Tth DNA Polymerases were obtained fromPromega Corporation (Madison, Wis.). Pfu (exo-) DNA Polymerase wasobtained from Stratagene, Inc. (La Jolla, Calif.). Phosphoramidites andcolumns for nucleic acid synthesis were obtained from Glen Research(Sterling, Va.).

Synthesis of a 3′-O-allyl modified 19-mer oligonucleotide

3′-O-allyl-thymidine phosphoramidite 3, prepared according to FIG. 1 wasused to synthesize a 19-mer oligonucleotide5′-AGA-GGA-TCC-AAC-CGA-GAC-T(allyl)-3′ 4 (MW=5871). The synthesis wascarried out in the 5′ to 3′ direction using 3 along with dA-5′-CE,dC-5′-CE, dG-5′-CE and dT-5′-CE phosphoramidites and a dA-5′-CPG column.The oligonucleotide was purified by HPLC using an Xterra MS C18 (4.6×50mm) column (Waters). The elution was performed over 90 min at a flowrate of 0.5 ml/min and a fixed temperature of 50° C. using a lineargradient (12-34.5%) of methanol in a buffer containing 8.6 mMtriethylamine and 100 mM hexafluoroisopropyl alcohol (pH=8.1). Theproduct was characterized using MALDI-TOF MS.

Deallylation reaction performed using the 3′-O-allyl modified 19-meroligonucleotide

For the deallylation reaction, 55 equivalents of Na₂PdCl₄ and 440equivalents of a trisodium triphenylphosphinetrisulfonate (TPPTS) ligandwere used in water at 70° C. Na₂PdCl₄ in degassed water (0.7 μl, 2.2nmol) was added to a solution of TPPTS in degassed water (1 μl, 17.6nmol) and mixed well. After 5 min, a solution of 3′-O-allyl modifiedoligonucleotide 4 (1 μl, 40 pmol) was added. The reaction mixture wasthen placed in a heating block at 70° C. and incubated for 30 seconds.The resulting deallylated product was desalted by Zip Tip (MilliporeCorporation) and analyzed using MALDI-TOF MS.

Primer Extension Reaction Performed with the Deallylated DNA Product

The 10 μl extension reaction mixture consisted of 45 pmol of thedeallylated DNA product as a primer, 100 pmol of a single-strandedsynthetic 100-mer DNA template (sequence shown in reference 15)corresponding to a portion of exon 7 of the p53 gene, 100 pmol ofBiotin-11-2′,3′-dideoxyguanosine-5′-triphosphate (Biotin-11-ddGTP)terminator (Perkin Elmer), 1× Thermo Sequenase reaction buffer and 4 Uof Thermo Sequenase DNA Polymerase. The extension reaction consisted of15 cycles at 94° C. for 20 sec, 48° C. for 30 sec and 60° C. for 60 sec.The product was purified using solid phase capture onstreptavidin-coated magnetic beads (25), desalted using Zip Tip andanalyzed using MALDI-TOF MS.

Synthesis of 3′-O-allyl-dUTP-PC-Bodipy-FL-510

3′-O-allyl-dUTP-PC-Bodipy-FL-510 10 was synthesized as shown in FIG. 2.Detailed synthesis procedures and characterization data for allintermediate compounds (6-9) are described in the supportinginformation.

PC-Bodipy-FL-510 NHS ester (13) (7.2 mg, 12 (mol) in 300 μl ofacetonitrile was added to a solution of3′-O-allyl-5-(3-aminoprop-1-ynyl)-2′-deoxyuridine-5′-triphosphate 9 (2mg, 4 (mol) in 300 μl of Na2CO3-NaHCO3 buffer (0.1 M, pH 8.7). Thereaction mixture was stirred at room temperature for 3 h. A preparativesilica-gel TLC plate was used to separate the unreacted PC-Bodipy-FL-510NHS ester from the fractions containing 10 (CHCl3/CH₃OH, 85/15). Theproduct was concentrated further under vacuum and purified withreverse-phase HPLC on a 150 (4.6-mm C18 column to obtain the pureproduct 10 (retention time=35 min). Mobile phase: A, 8.6 mMtriethylamine/100 mM hexafluoroisopropyl alcohol in water (pH 8.1); B,methanol. Elution was performed with 100% A isocratic over 10 minfollowed by a linear gradient of 0-50% B for 20 min and then 50% Bisocratic over another 20 min. 3′-O-allyl-dUTP-PC-Bodipy-FL-510 10 wascharacterized by the following single base extension reaction andMALDI-TOF MS.

Primer extension using 3′-O-allyl-dUTP-PC-Bodipy-FL-510 andphotocleavage of the extension product

An 18-mer oligonucleotide 5′-AGA-GGA-TCC-AAC-CGA-GAC-3′ (MW=5907) wassynthesized using dA-CE, dC-CE, dG-CE and Biotin-dT phosphoramidites. Aprimer extension reaction was performed using a 15 (1 reaction mixtureconsisting of 50 pmol of primer, 100 pmol of single-stranded synthetic100-mer DNA template corresponding to a portion of exon 7 of the p53gene (15), 200 pmol of 3′-O-allyl-dUTP-PC-Bodipy-FL-510, 1× Thermopolreaction buffer (New England Biolabs) and 15 U of 9 (N Polymerase (exo-)A485L/Y409V. The extension reaction consisted of 15 cycles of 94 (C for20 sec, 48 (C for 30 sec and 60 (C for 60 sec. A small portion of theDNA extension product 11 was desalted using Zip Tip and analyzed usingMALDI-TOF MS. The rest of the product was freeze-dried, resuspended in200 (1 of deionized water and irradiated for 10 sec in a quartz cellwith path lengths of 1.0 cm employing an Nd-YAG laser ((˜355 nm) tocleave the fluorophore from the DNA, yielding product 12.

Deallylation of the DNA extension product generated by the Incorporationof 3′-O-allyl-dUTP-PC-Bodipy-FL-510

The above photocleaved 3′-O-allyl modified DNA product 12 (180 pmolproduced in multiple reactions) was dried and resuspended in 1 (1 ofdeionized H2O. Na2PdCl4 in degassed H2O (4.1 (1, 72 nmol) was added to asolution of TPPTS in degassed H2O (2.7 (1, 9 nmol) and mixed well. After5 min, the above DNA product (1 (1, 180 pmol) was added. The reactionmixture was then placed in a heating block, incubated at 70° C. for 90sec to yield deallylated product 13, and then cooled to room temperaturefor analysis by MALDI-TOF MS.

Polymerase Extension and Photocleavage Using the Deallylated DNA Productas a Primer

The above deallylated DNA product 13 was used as a primer in a singlebase extension reaction. The 10 (1 reaction mixture consisted of 50 pmolof the above deallylated product 13, 125 pmol of dGTP-PC-Bodipy-FL-510(14), 4 U of Thermo Sequenase DNA Polymerase and 1× reaction buffer. Theextension reaction consisted of 15 cycles of 94 (C for 20 sec, 48 (C for30 sec and 60 (C for 60 sec. The DNA extension product 14 was desaltedusing the Zip Tip protocol and a small portion was analyzed usingMALDI-TOF MS. The remaining product was then irradiated with near UVlight for 10 sec to cleave the fluorophore from the extended DNAproduct. The resulting photocleavage product 15 was desalted andanalyzed using MALDI-TOF MS.

Discussion

It is shown here that an allyl moiety can be successfully used as ablocking group for the 3′-OH of a photocleavable fluorescent nucleotideanalogue in SBS to prevent the DNA polymerase reaction from continuingafter the incorporation of the 3′-O-allyl modified nucleotide analogue.Furthermore, it is demonstrated that the allyl group can be efficientlyremoved to generate a free 3′-OH group and allow the DNA polymerasereaction to continue to the subsequent cycle.

Conventional methods for cleavage of the allyl group combine atransition metal-catalyzed isomerization of the double bond to the enolether and subsequent hydrolysis of the latter to produce thecorresponding alcohol (26, 27). For application in SBS, it is importantto ensure that complete chemical cleavage of the 3′-O-allyl group can berapidly and specifically carried out while leaving the DNA intact.Trisodium triphenylphosphinetrisulfonate (TPPTS) has been widely used asa ligand for Pd mediated deallylation under aqueous conditions (28-30),while an active Pd catalyst can be generated from Na2PdCl4 and anappropriate ligand (31, 32). Thus, a water-soluble Pd catalyst systemgenerated from Na2PdCl4 and TPPTS was investigated for deallylation ofthe 3′-O-allyl modified DNA product.

To evaluate the cleavage conditions of the allyl group capping the 3′-OHof DNA, first a 19-mer oligonucleotide[5′-AGAGGATCCAACCGAGAC-T(allyl)-3′ ] was synthesized using3′-O-allyl-thymidine phosphoramidite (FIG. 3). The identity of thepurified oligonucleotide was established using MALDI-TOF massspectrometry. Then the above Na₂PdCl₄/TPPTS catalyst system was testedfor the deallylation of the oligonucleotide. In FIG. 3A, the mass peakat m/z 5871 corresponds to the mass of the purified oligonucleotidebearing the allyl group. FIG. 3B shows a single mass peak at m/z 5831indicating that near complete deallylation was achieved with aDNA/Na₂PdCl₄/TPPTS ratio of 1/55/440 in a reaction time of 30 seconds.The next step was to prove that the above deallylated DNA product couldbe used as a primer in a polymerase extension reaction. A single baseextension reaction was performed using the deallylated DNA product as aprimer, a synthetic template and a Biotin-11-ddGTP nucleotide terminatorwhich was complementary to the base immediately adjacent to the primingsite on the template. The DNA extension product was isolated using solidphase capture purification and analyzed using MALDI-TOF MS (25). Themass spectrum in FIG. 3C shows a clear peak at m/z 6535 corresponding tothe extension product indicating that the deallylated product can besuccessfully used as a primer in a polymerase reaction.

The above experiments established that Na₂PdCl₄ and TPPTS could be usedto efficiently carry out deallylation on DNA in an aqueous environment.Our next step was to investigate if a 3′-O-allyl-modified nucleotidecould be incorporated in a DNA polymerase reaction. For this purpose, anucleotide analogue 3′-O-allyl-thymidine triphosphate (3′-O-allyl-dTTP)was synthesized which was tested with 15 different polymerases forincorporation. The tested enzymes included Therminator, ThermoSequenase, Vent (exo-), Deep Vent (exo-), Tth, Tfl, Bst, Pfu (exo-),Klenow (exo-) fragment and Sequenase DNA Polymerases, AMV, RAV2, M-MulV,HIV reverse transcriptases and a 9°N Polymerase (exo-) bearing themutations A485L and Y409V. Our preliminary results showed that 9°N DNApolymerase (exo-) A485L/Y409V could efficiently incorporate3′-O-allyl-dTTP in an extension reaction, consistent with resultsreported recently (31).

After confirming the incorporation ability of 3′-O-allyl-dTTP into agrowing DNA strand by DNA polymerase, a new 3′-modified photocleavablefluorescent nucleotide analogue was synthesized,3′-O-allyl-dUTP-PC-Bodipy-FL-510, according to FIG. 2, and it wasestablished that the analogue can also can be efficiently incorporatedby the above polymerase. The aim was to evaluate that the presence ofthe bulky photocleavable fluorescent moiety on the base and the allylgroup on the 3′ end of the nucleotide analogue would not affect thepolymerase extension reaction. Furthermore, demonstration of an entirecycle of primer extension was desirable, photocleavage of thefluorophore, deallylation followed by extension with anotherphotocleavable fluorescent nucleotide complementary to the next base onthe template and photocleavage once again. This experiment will thustest the feasibility of using 3′-O-allyl-dUTP-PC-Bodipy-FL-510 as areversible terminator for SBS.

The entire cycle of a polymerase reaction using3′-O-allyl-dUTP-PC-Bodipy-FL-510 as a reversible terminator is depictedin FIG. 5. The extension product 11 obtained using3′-O-allyl-dUTP-PC-Bodipy-FL-510 and 9° N DNA Polymerase (exo-)A485L/Y409V was purified using HPLC and analyzed using MALDI-TOF MS. Thebase in the template immediately adjacent to the priming site was ‘A’.Thus, if 3′-O-allyl-dUTP-PC-Bodipy-FL-510 was accepted by the polymeraseas a terminator, the primer would extend by one base and then thereaction would terminate. Our results indicate that this was indeed thecase. After confirming that the extension reaction was successful, itwas irradiated with near UV light at 355 nm for 10 seconds to cleave thefluorophore from the DNA, generating product 12. In an SBS system, thisstep would ensure that there would be no carryover of the fluorescencesignal into the next incorporation cycle so as to prevent the generationof ambiguous data at each step, as shown in the accompanying paper (15).The photocleavage product 12 was then incubated with a Na₂PdCl₄/TPPTScatalyst system at 70° C. for 90 seconds to perform deallylation. Thedeallylated DNA product 13 was purified by reverse phase HPLC and thenused as a primer in a second DNA extension reaction to prove that theregenerated 3′-OH was capable of allowing the polymerase reaction tocontinue. For the extension reaction, a photocleavable fluorescentnucleotide dGTP-PC-Bodipy-FL-510 was used and Thermo Sequenase DNApolymerase. The extension product 14 was irradiated as above, for 10seconds to generate photocleavage product 15 and hence complete anentire reversible termination cycle.

After each step in the above cycle, a portion of the product waspurified and analyzed using MALDI-TOF MS to confirm its identity and thesuccessful completion of that step. Each product was desalted using theZip Tip desalting protocol to ensure the generation of sharp andwell-resolved data free from salt peaks. The MALDI-TOF MS data for eachstep are shown in FIG. 5. FIG. 5A shows the primer extension product 11at m/z 6787 generated using 3′-O-allyl-dUTP-PC-Bodipy-FL-510. The peakat m/z 6292 corresponds to the photocleavage product that was generatedby the partial photocleavage of the extension product due to thenitrogen laser (λ˜337 nm) used for ionization of the analyte inMALDI-TOF MS. FIG. 5B shows the photocleavage result after the 10-secondirradiation of the extension product at 355 nm. It can be seen from thedata that the peak at m/z 6787, corresponding to the extension producthas completely vanished and only a single peak corresponding to 12remains at m/z 6292, which proves that photocleavage was efficientlyachieved. FIG. 5C shows a similar single peak at m/z 6252, whichcorresponds to the deallylated photocleavage product 13. The absence ofa significant peak at m/z 6292 proves that deallylation was completedwith high efficiency. FIG. 5D shows the MALDI-TOF MS data for theextension product obtained using the above deallylated DNA product 13 asa primer and nucleotide analogue dGTP-PC-Bodipy-FL-510. A dominant peakis seen at m/z 7133 corresponding to the extension product 14. Finally,FIG. 5E shows a clear peak at m/z 6637 corresponding to thephotocleavage product 15 and no significant peak at m/z 7133 indicatingthat complete photocleavage had occurred.

The results of the above experiments provide sufficient proof of thefeasibility of using the allyl group as a reversible capping moiety forthe 3′-OH of the photocleavable nucleotide analogues for SBS. It isshown that a 3′-O-allyl modified nucleotide bearing a photocleavablefluorophore is an excellent substrate for 9°N DNA polymerase A485L/Y409Vand can be incorporated with high efficiency in a polymerase extensionreaction. It is also demonstrated that complete photocleavage isachieved in ˜10 seconds on these DNA products. Furthermore, it is shownthat deallylation can be swiftly achieved to near completion under mildreaction conditions in an aqueous environment using a palladiumcatalyst. Finally, it is have established that the deallylated DNAproduct can be used as a primer to continue the polymerase reaction andthat extension and photocleavage can be performed with high efficiency.These findings confirm that an allyl moiety protecting the 3′-OH groupindeed bestows the capability of reversible terminating abilities tophotocleavable nucleotide analogues, which can be used for SBS. Furtherefforts are being focused on generating four nucleotide analogues (A, C,G and T), each with a distinct photocleavable fluorophore and with a3′-O-allyl capping group. These nucleotides will facilitate thedevelopment of SBS for high-throughput DNA sequencing and genotypingapplications.

Example 2 Synopsis

DNA sequencing by synthesis (SBS) using reversible fluorescentnucleotide terminators is a potentially efficient approach to addressthe limitations of current DNA sequencing techniques. Here, the designand synthesis of a complete set of four-color 3′-O-allyl modifiedphotocleavable fluorescent nucleotides as reversible terminators for SBSis described. The nucleotides are efficiently incorporated by DNApolymerase into a growing DNA strand to terminate the polymerasereaction. After that the fluorophore is photocleaved quantitatively byirradiation at 355 nm, and the allyl group is rapidly and efficientlyremoved by using a Pd-catalyzed reaction under DNA compatible conditionsto regenerate a free 3′-OH group to reinitiate the polymerase reaction.A homopolymeric region of a DNA template was successfully sequencedusing these 3′-O-allyl modified nucleotide analogues, facilitating thedevelopment of SBS as a viable approach for high-throughput DNAsequencing

Introduction

The design and synthesis of a complete set of four-color 3′-O-allylmodified photocleavable fluorescent nucleotides,3′-O-allyl-dGTP-PC-Bodipy-FL-510, 3′-O-allyl-dCTP-PC-Bodipy-650,3′-O-allyl-dUTP-PC-R6G and 3′-O-allyl-dATP-PC-ROX, is disclosed here, asshown in FIG. 6. Their applications as reversible terminators for SBSare also described here, demonstrating the base-specific incorporationof these nucleotide analogues by DNA polymerase, the highly efficientphotocleavage of the fluorescent dye, and the rapid and complete removalof 3′-O-allyl group in a Pd-catalyzed reaction under DNA compatibleconditions. Previously, the Pd-catalyzed deallylation to regenerate afree 3′-OH of the DNA extension product was carried out in pure water(34) which can destabilize the primer-template duplex. A new conditionfor rapid quantitative deallylation in a buffer solution at pH 8.8 hasbeen identified here, which is commonly used in a polymerase reaction.The successful synthesis of these 3′-O-allyl modified photocleavablefluorescent nucleotides as reversible terminators to sequence through ahomopolymer sequence, and the identification of the new deallylationcondition will facilitate the development of SBS as a viable approachfor de novo DNA sequencing.

As an example, 3′-O-allyl-dGTP-PC-Bodipy-FL-510 (10) is used here toillustrate the detailed synthesis strategy and procedures. To theapplicants' knowledge, using 3′-modified dGTP as a reversible terminatorfor SBS has not been reported, partly due to the difficulty of modifying3′-OH of guanosine by a suitable capping group without protecting theguanine base. Structure 10 was prepared following a synthesis route asshown in FIG. 7.

2-amino-4-methoxy-7-(β-D-2-deoxyribofuranosyl) pyrrolo[2,3-d]-pyrimidine1 was chosen as the starting material for the synthesis of3′-O-allyl-dGTP 9 (FIG. 7) (see (35)). Structure 1 was first protectedby isobutyryl chloride to form 2 quantitively (4). Structure 2 wasiodinized with NIS to afford a single iodo product 3 in 84% yield, asthe protected 2-amino group in the heterocyclic ring promotes theformation of 7-substituted product (5). Structure 3 was deprotected toafford 4 in 91% yield by sodium methoxide. The 5′-OH of 4 was protectedby tert-butyldimethylsilyl chloride to yield 5 in 88% yield (6). The3′-OH of 5 was subsequently allylated in CH₂Cl₂ and 40% aqueous NaOHsolution using tetrabutylammonium bromide as phase-transfer catalyst togive a 92% yield of 6 without 2-N-allylated product. Cross-couplingreaction of 6 with the terminal alkyne catalyzed by Pd(0)/Cu(I) formed 7in 94% yield (7). Next, a one-pot procedure of demethylation (8) anddesilylation of 7 gave a moderate 34% yield of 8. Finally structure 8was transformed into the corresponding triphosphate 9 followingestablished procedures (9). Coupling 9 with PC-Bodipy-FL-510 NHS ester(2) yielded the target compound, 3′-O-allyl-dGTP-PC-Bodipy-FL-510,structure 10.

3′-O-allyl-dATP-PC-ROX 19 was also prepared, as were3′-O-allyl-dCTP-PC-Bodipy-650 26 and 3′-O-allyl-dUTP-PC-R6G 33, as shownin FIGS. 8, 9, and 10, respectively.

For 3′-O-allyl modified PC fluorescent nucleotides to act as reversibleterminators for SBS, it is important to establish that they can be usedto determine a repeated DNA sequence in a polymerase reaction. To thisend, polymerase DNA extension reactions were performed using thesenucleotides as substrates in solution. This allows the isolation of theDNA product at each step of SBS for detailed molecular structurecharacterization by using MALDI-TOF mass spectrometry (MS).

3′-O-allyl-dGTP-PC-Bodipy-FL-510 (structure 10) was used as a substratein a DNA extension reaction as shown in FIG. 11. A synthetic 100-mer DNAcorresponding to a portion of exon 7 of the human p53 gene was used as atemplate to perform the extension. The sequence in the templateimmediately adjacent to the annealing site of the primer had a repeatingsequence of 3′-CC-5′. First, a polymerase extension reaction using 10 asa terminator along with a primer and the above template was performed.After the reaction, a small portion of the DNA extension product wascharacterized by MALDI-TOF MS. The rest of the product was irradiated at355 nm for 10 sec to cleave the fluorophore from the DNA and thenanalyzed by MALDI-TOF MS. After photocleavage, the DNA product was addedto a deallylation cocktail [1× Thermopol reactionbuffer/Na₂PdCl₄/—P(PhSO₃Na)₃] to remove the 3′-Allyl group in 30 sec toyield quantitatively deallylated DNA product. The deallylated DNAproduct with a free 3′-OH group regenerated was then used as a primer toincorporate 10 in a subsequent second polymerase extension reaction.

FIG. 12 (right panel) shows sequential mass spectrum at each step of DNAsequencing by synthesis using 10 as a reversible terminator. As can beseen from FIG. 12, panel (A), the MALDI-TOF MS spectrum consists of adistinct peak at m/z 7,052 corresponding to the single base DNAextension product 34 with 100% incorporation efficiency, confirming thatthe reversible terminator 10 can be incorporated base-specifically byDNA polymerase into a growing DNA strand. The small peak at m/z 6,556corresponding to the photocleavage product is due to the partialcleavage caused by the nitrogen laser pulse (337 nm) used in MALDIionization. FIG. 12, panel (B) shows the photocleavage result after 10sec irradiation of the DNA extension product at 355 nm. The peak at m/z7,052 has completely disappeared, whereas the peak corresponding to thephotocleavage product 35 appears as the sole dominant peak at m/z 6,556.FIG. 12, panel (C) shows a single peak at m/z 6,516, which correspondsto a deallylated photocleavage product 36. The absence of a peak at m/z6,556 proves that the deallylation reaction was completed with highefficiency. The next extension reaction was carried out by using thisdeallylated photocleavage product 36 as a primer along with3′-O-allyl-dGTP-PC-Bodipy-FL-510 (10) to yield an extension product 37(FIG. 12, panel D). DNA products (38 and 39) from photocleavage (FIG.12, panel E) and deallylation (FIG. 12, panel F) respectively wereobtained in similar manner as described previously, thereby completingtwo entire polymerase extension cycles to sequence a homopolymericregion of a template using 10 as a reversible terminator.

3′-O-allyl-dATP-PC-ROX 19, mixed together with3′-O-allyl-dGTP-PC-Bodipy-FL-510 10/3′-O-allyl-dCTP-PC-Bodipy-65026/3′-O-allyl-dUTP-PC-R6G 33, was used as a reversible terminator in aprimer extension reaction as shown in FIG. 13 (left panel). After theincorporation, photocleavage and deallylation reactions were performedon the DNA extension product, following a similar procedure as for 10.FIG. 13 (right panel, A) shows the MOLDI-TOF MS results for thecharacterization of the product from each step. In the extensionreaction, all four 3′-O-allyl modified photocleavable fluorescentnucleotides were added simultaneously instead of using only the correctone. The MS showed that only 3′-O-allyl-dATP-PC-ROX 19, the onecomplementary with the template sequence, was successfully incorporatedin this extension reaction, as demonstrated by the single major peak atm/z 7,228 and a partial photocleavage peak at m/z 6,495. There is noother DNA extension product observed, indicating a faithfulincorporation of the 3′-O-allyl modified nucleotide. The MS results alsodemonstrated that the photocleavage and deallylation steps weresuccessfully conducted as shown in FIG. 13 (right panel, B and C) withpeaks at m/z 6,495 and 6,455.

3′-O-allyl-dCTP-PC-Bodipy-650 26, mixed together with3′-O-allyl-dGTP-PC-Bodipy-FL-510 10/3′-O-allyl-dATP-PC-ROX19/3′-O-allyl-dUTP-PC-R6G 33, was used in a primer extension reactionand then photocleavage and deallyation reactions were performed on theDNA extension product, as shown in FIG. 14 (left panel). FIG. 14 (rightpanel, A) shows the successful incorporation of 26, among the fournucleotide analogues, by the DNA polymerase to generate a single DNAextension product 43 at m/z 8,532. Subsequently, photocleavage wasconducted to generate a photocleavage product 44 at m/z 7,762, anddeallyation product 45 was observed at m/z 7,722, as shown in FIG. 14(right panel, B and C), respectively.

Similarly, 3′-O-allyl-dUTP-PC-R6G 33, mixed together with3′-O-allyl-dGTP-PC-Bodipy-FL-510 10/3′-O-allyl-dATP-PC-ROX19/3′-O-allyl-dCTP-PC-Bodipy-650 26, also showed successfulincorporation by a DNA polymerase in a primer extension reaction, asindicated by the single extension product (46) peak at m/z 6,210 inMALDI-TOF MS spectrum in FIG. 15 (right panel, A). The fluorescent dyewas then photocleaved to generate a photocleavage product 47 at m/z5,552, and 3′-O-allyl was removed in a Pd-catalyzed reaction to generatea deallylated product 48 at m/z 5,512, as shown in FIG. 15 (right panel,B and C), respectively.

Material and Methods General Information

1H NMR spectra were recorded on Brucker DPX-400 (400 MHz) and BruckerDPX-300 spectrometers and are reported in ppm from CD₃OD or DMSO-d6internal standard (3.31 or 2.50 ppm respectively). Data are reported asfollows: (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet,dd=doublet of doublets, ddd=doublet of doublets of doublets; couplingconstant(s) in Hz; integration; assignment). Proton decoupled ¹³C NMRspectra were recorded on a Brucker DPX-400 (100 MHz) spectrometer andare reported in ppm from CD₃OD, DMSO-d6, or CDCl₃ internal standard(49.0, 39.5, or 77.0 ppm respectively). Proton decoupled ³¹P NMR spectrawere recorded on a Brucker DPX-300 (121.4 MHz) spectrometer withoutcalibration. High Resolution Mass Spectra (HRMS) were obtained on a JEOLJMS HX 110A mass spectrometer. Mass measurement of DNA was made on aVoyager DE MALDI-TOF mass spectrometer (Applied Biosystems). Photolysiswas performed by using a Spectra Physics GCR-150-30 Nd-yttrium/aluminumgarnet laser that generates light pulses at 355 nm. Compounds 1 and 11were purchased from Berry & Associates (Dexter, Mich.). Bodipy-FL-510NHS ester, ROX NHS ester, Bodipy-650 NHS ester and R6G NHS ester werepurchased from Invitrogen (Carlsbad, Calif.). All other chemicals werepurchased from Sigma-Aldrich. 9° N polymerase (exo-) A485L/Y409V wasgenerously provided by New England Biolabs.

I. Synthesis of 3′-O-allyl modified photocleavable fluorescentnucleotides 1) Synthesis of 3′-O-allyl-dGTP-PC-Bodipy-FL-510 as shown inFIG. 72-(2-Methylpropanoyl)amino-7-[3′,5′-bis-O-(2-methylpropanoyl)-β-D-2′-deoxyribofuranosyl]-4-methoxypyrrolo[2,3-d]pyrimidine(2)

To a stirred suspension of 1 (1.00 g; 3.57 mmol) in anhydrous pyridine(35 mL) was added slowly isobutyryl chloride (3.40 mL; 32.2 mmol) at 0°C. The reaction mixture was stirred at 0° C. for 1 h. Methanol (2 mL)was then added and the reaction mixture was stirred for another 10 min.Then most solvent was removed under vacuum. Ethyl acetate (200 mL) andsaturated aqueous NaHCO₃ (50 mL) were added to the residue. The organiclayer was separated and washed by saturated aqueous NaHCO₃ and NaClrespectively, and dried over anhydrous Na₂SO₄. After evaporation of thesolvent, the residue was purified by flash column chromatography oversilica gel using ethyl acetate-hexane (1:3-2) as the eluent to afford 2as white foam (1.75 g; 99% yield): ¹H NMR (400 MHz, CD₃OD) δ 7.28 (d,J=3.7 Hz, 1H, 6-H), 6.66 (dd, J=5.9, 8.6 Hz, 1H, 1′-H), 6.51 (d, J=3.7Hz, 1H, 5-H), 5.41 (m, 1H, 3′-H), 4.33-4.36 (m, 2H, 5′-H), 4.22 (m, 1H,4′-H), 4.08 (s, 3H, 4-OCH₃), 2.83-2.96 (m, 2H, one of CH(CH₃)₂ and oneof 2′-H), 2.54-2.70 (m, 2H, two of CH(CH₃)₂), 2.48-2.54 (ddd, J=2.0,5.9, 14.2 Hz, one of 2′-H), 1.15-1.23 (m, 18H, CH(CH₃)₂); ¹³C NMR (100MHz, CD₃OD) δ 178.2, 177.7, 177.4, 164.2, 153.4, 152.5, 123.4, 103.5,100.7, 85.2, 83.0, 75.9, 65.0, 54.4, 37.9, 36.6, 35.0, 34.9, 19.9 (twoCH₃), 19.3-19.4 (four peaks for four CH₃); HRMS (FAB+) calcd forC₂₄H₃₅O₇N₄ (M+H⁺): 491.2506. found: 491.2503.

2-(2-Methylpropanoyl)amino-7-[3′,5′-bi-O-(2-methylpropanoyl)-β-D-2′-deoxyribo-furanosyl]-5-iodo-4-methoxypyrrolo[2,3-d]pyrimidine(3)

To a vigorously stirred solution of 2 (1.75 g; 3.57 mmol) in anhydrousDMF (27 mL) was added 95% N-iodosuccimide (NIS) (866 mg; 3.66 mmol). Thereaction mixture was stirred at room temperature for 22 h, and then mostsolvent was removed under vacuum. Diethyl ether (200 mL) and saturatedaqueous NaHCO₃ (50 mL) were added. The organic layer was separated andwashed by saturated aqueous NaCl, and dried over anhydrous Na₂SO₄. Afterevaporation of the solvent, the residue was purified by flash columnchromatography over silica gel using ethyl acetate-hexane (1:3) as theeluent to afford 3 as white solid (1.98 g; 90% yield): ¹H NMR (400 MHz,CD₃OD) δ 7.43 (s, 1H, 6-H), 6.63 (dd, J=6.0, 8.2 Hz, 1H, 1′-H), 5.41 (m,1H, 3′-H), 4.33-4.36 (m, 2H, 5′-H), 4.23 (m, 1H, 4′-H), 4.09 (s, 3H,4-OCH₃), 2.78-2.94 (m, 2H, one of CH(CH₃)₂ and one of 2′-H), 2.57-2.70(m, 2H, two of CH(CH₃)₂), 2.50-2.57 (ddd, J=2.3, 6.0, 14.2 Hz, one of2′-H), 1.17-1.24 (m, 18H, CH(CH₃)₂); ¹³C NMR (100 MHz, CD₃OD) δ 178.3,177.8, 177.5, 164.3, 153.3, 152.8, 128.6, 105.2, 85.3, 83.3, 75.8, 65.0,54.4, 51.8, 38.2, 36.8, 35.2, 35.1, 19.9 (two CH₃), 19.3-19.5 (fourpeaks for four CH₃); HRMS (FAB+) calcd for C₂₄H₃₄O₇N₄I (M+H⁺): 617.1472.found: 617.1464.

2-Amino-7-(β-D-2′-deoxyribofuranosyl)-5-iodo-4-methoxypyrrolo[2,3-d]pyrimidine(4)

3 (1.98 g; 3.21 mmol) was dissolved in 0.5 M methanolic CH₃ONa (50 mL)and stirred at 65° C. for 12 h. Saturated aqueous NaHCO₃ (20 mL) wasadded and the mixture was stirred for 10 min. Then most of methanol wasevaporated and the residue was extracted by ethyl acetate (150 mL). Theorganic layer was washed by saturated aqueous NaHCO₃ and NaClrespectively, and dried over anhydrous Na₂SO₄. After evaporation of thesolvent, the residue was purified by flash column chromatography oversilica gel using CH₃OH—CH₂Cl₂ (1:30-15) as the eluent to afford 4 aswhite solid (1.23 g; 94% yield): ¹H NMR (400 MHz, CD₃OD) δ 7.17 (s, 1H,6-H), 6.36 (dd, J=6.0, 8.4 Hz, 1H, 1′-H), 4.47 (m, 1H, 3′-H), 3.99 (s,3H, 4-OCH₃), 3.96 (m, 1H, 4′-H), 3.77 (dd, J=3.4, 12.0 Hz, 1H, one of5′-H), 3.70 (dd, J=3.7, 12.0 Hz, 1H, one of 5′-H), 2.55-2.64 (ddd,J=6.0, 8.4, 13.4 Hz, one of 2′-H), 2.20-2.26 (ddd, J=2.4, 5.9, 13.4 Hz,one of 2′-H); ¹³C NMR (100 MHz, CD₃OD) δ 164.7, 160.6, 154.3, 126.5,101.6, 88.7, 86.0, 73.0, 63.7, 53.7, 51.3, 41.1; HRMS (FAB+) calcd forC₁₂H₁₆O₄N₄I (M+H⁺): 407.0216. found: 407.0213.

2-Amino-7-[β-D-5′-O-(tert-butyldimethylsilyl)-2′-deoxyribofuanosyl]-5-iodo-4-methoxypyrrolo[2,3-d]pyrimidine(5)

To a stirred solution of 4 (1.23 g; 3.02 mmol) and imidazole (494 mg;7.24 mmol) in anhydrous DMF (15 mL) was added tert-butyldimethylsilylchloride (TBDMSCl) (545 mg; 3.51 mmol). The reaction mixture was stirredat room temperature for 20 h. Then most solvent was removed undervacuum, and the residue was purified by flash column chromatography oversilica gel using ethyl acetate-hexane (1:2˜0.5) as the eluent to afford5 as white foam (1.38 g; 88% yield): ¹H NMR (400 MHz, CD₃OD) δ 7.23 (s,1H, 6-H), 6.49 (dd, J=6.1, 7.7 Hz, 1H, 1′-H), 4.46 (m, 1H, 3′-H), 3.99(s, 3H, 4-OCH₃), 3.94 (m, 1H, 4′-H), 3.79-3.87 (m, 2H, 5′-H), 2.36-2.44(ddd, J=5.8, 7.7, 13.3 Hz, one of 2′-H), 2.24-2.31 (ddd, J=3.1, 6.0,13.3 Hz, one of 2′-H), 0.96 (s, 9H, C(CH₃)₃), 0.14 (s, 3H, one ofSiCH₃), 0.13 (s, 3H, one of SiCH₃); ¹³C NMR (100 MHz, CD₃OD) δ 164.6,160.7, 154.7, 125.1, 101.0, 88.2, 84.2, 72.7, 64.7, 53.7, 51.7, 41.9,26.7, 19.4, −5.0, −5.1; HRMS (FAB+) calcd for C₁₈H₃₀O₄N₄SiI (M+H⁺):521.1081. found: 521.1068.

7-[β-D-3′-O-Allyl-5′-O-(tert-butyldimethysilyl)-2′-deoxyribofuranosyl]-2-amino-5-iodo-4-methoxypyrrolo[2,3-d]pyrimidine(6)

To a stirred solution of 5 (1.38 g; 2.66 mmol) in CH₂Cl₂ (80 mL) wereadded tetrabutylammonium bromide (TBAB) (437 mg; 1.33 mmol), allylbromide (1.85 mL, 21.4 mmol) and 40% aqueous NaOH solution (40 mL). Thereaction mixture was stirred at room temperature for 1 h. Ethyl acetate(200 25 mL) was added and the organic layer was separated. The aqueouslayer was extracted with ethyl acetate (2×50 mL). The combined organiclayer was washed by saturated aqueous NaHCO₃ and NaCl respectively, anddried over anhydrous Na₂SO₄. After evaporation of the solvent, theresidue was purified by flash column chromatography over silica gelusing ethyl acetate-hexane (1:3) as the eluent to afford 6 as whitesolid (1.37 g; 92% yield): ¹H NMR (400 MHz, CD₃OD) δ 7.20 (s, 1H, 6-H),6.43 (dd, J=6.2, 7.9 Hz, 1H, 1′-H), 5.89-5.99 (m, 1H, CH₂CH═CH₂),5.29-5.35 (dm, J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.16-5.21 (dm, J=10.5Hz, 1H, one of CH₂CH═CH₂), 4.24 (m, 1H, 3′-H), 4.01-4.11 (m, 3H, 4′-Hand CH₂CH═CH₂), 3.99 (s, 3H, 4-OCH₃), 3.76-3.84 (m, 2H, 5′-H), 2.32-2.44(m, 2H, 2′-H), 0.95 (s, 9H, C(CH₃)₃)₃), 0.14 (s, 3H, one of SiCH₃), 0.13(s, 3H, one of SiCH₃); ¹³C NMR (100 MHz, CDCl₃) δ 163.3, 158.6, 153.6,134.1, 123.7, 116.9, 100.6, 84.4, 83.0, 79.1, 70.0, 63.6, 53.3, 51.1,38.1, 26.1, 18.5, −5.1, −5.3; HRMS (FAB+) calcd for C₂₁H₃₄O₄N₄SiI(M+H⁺): 561.1394. found: 561.1390.

7-[β-D-3′-O-Allyl-5′-O-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-2-amino-5-[3-[(trifluoroacetyl)amino]-prop-1-ynyl]-4-methoxypyrrolo[2,3-d]pyrimidine(7)

To a stirred solution of 6 (1.37 g; 2.45 mmol) in anhydrous DMF (11 mL)were added tetrakis(triphenylphosphine)palladium(0) (286 mg; 0.245 mmol)and CuI (101 mg; 0.532 mmol). The solution was stirred at roomtemperature for 10 min. Then N-propargyltrifluoroacetamide (1.12 g; 7.43mmol) and triethylamine (0.68 mL; 4.90 mmol) were added. The reactionwas stirred at room temperature for 13 h with exclusion of air andlight. Most DMF was removed under vacuum and the residue was dissolvedin ethyl acetate (100 mL). The solution was washed by saturated aqueousNaHCO₃ and NaCl respectively, and dried over anhydrous Na₂SO₄. Afterevaporation of the solvent, the residue was purified by flash columnchromatography over silica gel using ethyl acetate-hexane (1:31.5) andCH₃OH—CH₂Cl₂ (1:30) respectively as the eluent to afford 7 as yellowsolid (1.34 g; 94% yield): ¹H NMR (400 MHz, CD₃OD) δ 7.34 (s, 1H, 6-H),6.42 (dd, J=6.2, 7.7 Hz, 1H, 1′-H), 5.88-5.99 (m, 1H, CH₂CH═CH₂),5.28-5.35 (dm, J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.16-5.21 (dm, J=10.5Hz, 1H, one of CH₂CH═CH₂), 4.29 (s, 2H, C≡CCH₂), 4.24 (m, 1H, 3′-H),4.00-4.09 (m, 3H, 4′-H and CH₂CH═CH₂), 3.98 (s, 3H, 4-OCH₃), 3.76-3.84(m, 2H, 5′-H), 2.32-2.45 (m, 2H, 2′-H), 0.94 (s, 9H, C(CH₃)₃), 0.12 (s,3H, one of SiCH₃), 0.11 (s, 3H, one of SiCH₃); ¹³C NMR (100 MHz, CD₃OD)δ 165.0, 161.2, 158.1 (q, J=36 Hz, COCF₃), 154.2, 135.6, 125.0, 117.2(q, J=284 Hz, COCF₃), 117.0, 99.2, 97.3, 86.0, 84.6, 84.5, 80.3, 78.0,71.0, 64.8, 53.8, 39.0, 30.9, 26.5, 19.3, −5.1, −5.2; HRMS (FAB+)calculated for C₂₆H₃₇O₅N₅F₃Si (M+H⁺): 584.2516. found: 584.2491.

3′-O-Allyl-7-deaza-7-[3-[(trifluoroacetyl)amino]-prop-1-ynyl]-2′-deoxyguanosine(8)

To a stirred solution of 7 (1.34 g; 2.30 mmol) in anhydrous CH₃CN (86mL) were added NaI (363 mg; 2.42 mmol) and chlorotrimethylsilane (TMSCl)(0.306 mL; 2.42 mmol). The reaction was stirred at room temperature for1 h and then at 50° C. for 12 h. The solvent was evaporated and theresidue was dissolved in anhydrous THF (76 mL). 1 M tetrabutylammoniumfluoride (TBAF) in THF solution (4.80 mL; 4.80 mmol) was added and thereaction was stirred at room temperature for 1 h. The solvent wasevaporated and the residue was dissolved in ethyl acetate (150 mL). Thesolution was washed by saturated aqueous NaCl and dried over anhydrousNa₂SO₄. After evaporation of the solvent, the residue was purified byflash column chromatography over silica gel using CH₃OH-ethyl acetate(1:30) as the eluent to afford 8 as yellow solid (356 mg; 34% yield): ¹HNMR (400 MHz, CD₃OD) δ 7.21 (s, 1H, 6-H), 6.30 (dd, J=6.0, 8.4 Hz, 1H,1′-H), 5.88-5.99 (m, 1H, CH₂CH═CH₂), 5.28-5.35 (dm, J=17.3 Hz, 1H, oneof CH₂CH═CH₂), 5.15-5.20 (dm, J=10.5 Hz, 1H, one of CH₂CH═CH₂), 4.29 (s,2H, C≡CCH₂), 4.23 (m, 1H, 3′-H), 4.00-4.10 (m, 3H, 4′-H and CH₂CH═CH₂),3.65-3.75 (m, 2H, 5′-H), 2.41-2.49 (ddd, J=5.8, 8.4, 13.6 Hz, 1H, one of2′-H), 2.34-2.40 (ddd, J=2.3, 6.0, 13.6 Hz, 1H, one of 2′-H); ¹³C NMR(100 MHz, CD₃OD) δ 160.9, 158.0 (q, J=36 Hz, COCF₃), 154.1, 151.8,135.6, 124.4, 117.2 (q, J=284 Hz, COCF₃), 117.0, 101.4, 99.7, 86.4,85.5, 84.8, 80.7, 78.0, 71.0, 63.7, 38.5, 31.2; HRMS (FAB+) calcd forC₁₉H₂₁O₅N₅F₃ (M+H⁺): 456.1495. found: 456.1493.

3′-O-Allyl-7-deaza-7-(3-aminoprop-1-ynyl)-2′-deoxyguanosine-5′-triphosphate(9)

The procedure is the same as that of preparing3′-O-allyl-5-(3-aminoprop-1-ynyl)-2′-deoxyuridine-5′-triphosphate inRef. 34a to yield 9 as colorless syrup: ¹H NMR (300 MHz, D₂O) δ 7.56 (s,1H, 6-H), 6.37 (t, J=7.3 Hz, 1H, 1′-H), 5.89-6.02 (m, 1H, CH₂CH═CH₂),5.31-5.39 (dm, J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.21-5.28 (dm, J=10.5Hz, 1H, one of CH₂CH═CH₂), 4.49 (s, 2H, C≡CCH₂), 4.32 (m, 1H, 3′-H),4.06-4.18 (m, 3H, 4′-H and CH₂CH═CH₂), 3.92-3.99 (m, 2H, 5′-H),2.44-2.60 (m, 2H, 2′-H); ³¹P NMR (121.4 MHz, D₂O) δ −6.1 (d, J=20.8 Hz,1P, γ-P), −10.8 (d, J=18.9 Hz, IP, α-P), −21.9 (t, J=19.8 Hz, 1P, β-P).

3′-O-Allyl-dGTP-PC-Bodipy-FL-510 (10)

PC-Bodipy-FL-510 NHS ester (prepared by the same procedure in Ref. 34a)(7.2 mg, 12 μmol) in 300 μL of acetonitrile was added to a solution of 9(2 mg, 3.4 μmol) in 300 μL of Na₂CO₃—NaHCO₃ aqueous buffer (0.1 M, pH8.5). The reaction mixture was stirred at room temperature for 3 h. Apreparative silica-gel TLC plate was used to separate the unreactedPC-Bodipy-FL-510 NHS ester from the fraction containing 10 withCHCl₃—CH₃OH (85:15) as the eluent. The product was concentrated furtherunder vacuum and purified with reverse-phase HPLC on a 150×4.6-mm C18column to obtain the pure product 10 (retention time of 34 min). Mobilephase: A, 8.6 mM triethylamine/100 mM hexafluoroisopropyl alcohol inwater (pH 8.1); B, methanol. Elution was performed with 100% A isocraticover 10 min, followed by a linear gradient of 0-50% B for 20 min andthen 50% B isocratic over another 20 min.3′-O-allyl-dGTP-PC-Bodipy-FL-510 10 was characterized by primerextension reaction and MALDI-TOF MS.

2) Synthesis of 3′-O-Allyl-dATP-PC-ROX as shown in FIG. 84-Chloro-5-iodopyrrolo[2,3-d]pyrimidine (12)

To a vigorously stirred solution of 11 (1.0 g; 6.51 mmol) in CH₂Cl₂ (55mL) was added 95% N-iodosuccimide (1.70 g; 7.18 mmol). The reactionmixture was stirred at room temperature for 1 h, during which time moreprecipitate appeared. The solid was filtered and recrystallized in hotmethanol to afford 12 as slightly grey crystals (1.49 g; 82% yield): ¹HNMR (400 MHz, DMSO-d6) δ 12.96 (s br, 1H, NH), 8.59 (s, 1H, 2-H), 7.94(s, 1H, 6-H); ¹³C NMR (100 MHz, DMSO-d6) δ 151.2, 150.4, 150.2, 133.6,115.5, 51.7; HRMS (FAB+) calcd for C₆H₄N₃ClI (M+H⁺): 279.9139. found:279.9141.

4-Chloro-7-β-D-2′-deoxyribofuranosyl)-5-iodopyrrolo[2,3-d]pyrimidine(13)

To a stirred solution of 12 (597 mg; 2.14 mmol) in CH₃CN (36 mL) wereadded KOH powder (0.30 g; 5.36 mmol) andtris[2-(2-methoxyethoxy)ethyl]amine (44 μL, 0.14 mmol). The mixture wasstirred at room temperature for 10 min and then 90% 3,5-di-O-(p-toluyl)-2-deoxy-D-ribofuranosyl chloride (1.00 g; 2.31 mmol)was added. The reaction was stirred vigorously at room temperature for 1h, and the insoluble material was filtered and washed by hot acetone.The combined solution was evaporated and dissolved in 7M methanolicammonia (72 mL). The solution was stirred at room temperature for 24 h.After evaporation of the solvent, the residue was purified by flashcolumn chromatography over silica gel using CH₃OH—CH₂Cl₂ (0˜1:20) as theeluent to afford 13 as white solid (711 mg; 84% yield): 1H NMR (400 MHz,CD₃OD) δ 8.57 (s, 1H, 2-H), 8.08 (s, 1H, 6-H), 6.72 (dd, J=6.3, 7.5 Hz,1H, 1′-H), 4.53 (m, 1H, 3′-H), 4.00 (m, 1H, 4′-H), 3.80 (dd, J=3.6, 12.0Hz, 1H, one of 5′-H), 3.74 (dd, J=3.6, 12.0 Hz, 1H, one of 5′-H),2.56-2.64 (ddd, J=6.1, 7.5, 13.5 Hz, 1H, one of 2′-H), 2.36-2.43 (ddd,J=3.3, 6.2, 13.5 Hz, 1H, one of 2′-H); ¹³C NMR (100 MHz, CD₃OD) δ 152.9,151.7, 151.3, 134.7, 118.5, 89.0, 85.7, 72.6, 63.2, 52.6, 41.7; HRMS(FAB+) calcd for C₁₁H₁₂O₃N₃ClI (M+H⁺): 395.9612. found: 395.9607.

7-[β-D-5′-O-(tert-Butyldimethylilyl)-2′-deoxyribofuranosyl]-4-chloro-5-iodopyrro-lo[2,3-d]pyrimidine(14)

The procedure is the same as that of 5 and the crude was purified byflash column chromatography over silica gel using ethyl acetate-hexane(1:32) as the eluent to afford 14 as white solid (65% yield) and 30% ofthe starting material 13: ¹H NMR (400 MHz, CD₃OD) δ 8.56 (s, 1H, 2-H),7.99 (s, 1H, 6-H), 6.73 (t, J=6.7 Hz, 1H, 1′-H), 4.52 (m, 1H, 3′-H),4.02 (m, 1H, 4′-H), 3.92 (dd, J=3.0, 11.4 Hz, 1H, one of 5′-H), 3.86(dd, J=3.1, 11.4 Hz, 1H, one of 5′-H), 2.47-2.55 (ddd, J=5.8, 7.1, 13.4Hz, 1H, one of 2′-H), 2.40-2.47 (ddd, J=3.6, 6.3, 13.4 Hz, 1H, one of2′-H), 0.94 (s, 9H, C(CH₃)₃), 0.14 (s, 3H, one of SiCH₃), 0.13 (s, 3H,one of SiCH₃); ¹³C NMR (100 MHz, CD₃OD) δ 152.8, 151.5, 151.3, 133.8,118.2, 88.9, 85.4, 72.5, 64.6, 52.6, 42.4, 26.7, 19.5, −4.9, −5.0; HRMS(FAB+) calcd for C₁₇H₂₆O₃N₃ClSiI (M+H⁺): 510.0477. found: 510.0487.

7-[β-D-3′-O-Allyl-5′-O-(tert-butyldimethylsilyl)-2′-deoxyribofuranosyl]-4-chloro-5-iodopyrrolo[2,3-d]pyrimidine(15)

The procedure is the same as that of 6 and the crude was purified byflash column chromatography over silica gel using ethyl acetate-hexane(1:6) as the eluent to afford 15 as yellow oil (752 mg; 95% yield): 1HNMR (400 MHz, CD₃OD) δ 8.52 (s, 1H, 2-H), 7.94 (s, 1H, 6-H), 6.64 (dd,J=6.1, 7.6 Hz, 1H, 1′-H), 5.88-5.99 (m, 1H, CH₂CH═CH₂), 5.28-5.34 (dm,J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.16-5.21 (dm, J=10.4 Hz, 1H, one ofCH₂CH═CH₂), 4.28 (m, 1H, 3′-H), 4.13 (m, 1H, 4′-H), 4.01-4.11 (m, 2H,CH₂CH═CH₂), 3.88 (dd, J=3.6, 11.2 Hz, 1H, one of 5′-H), 3.80 (dd, J=3.1,11.3 Hz, 1H, one of 5′-H), 2.51-2.57 (ddd, J=2.7, 6.0, 13.5 Hz, 1H, oneof 2′-H), 2.42-2.50 (ddd, J=5.7, 7.7, 13.5 Hz, 1H, one of 2′-H), 0.93(s, 9H, C(CH₃)₃), 0.13 (s, 3H, one of SiCH₃), 0.12 (s, 3H, one ofSiCH₃); ¹³C NMR (100 MHz, CD₃OD) δ 152.8, 151.4, 151.3, 135.5, 133.6,118.2, 117.2, 86.5, 85.6, 80.2, 71.0, 64.8, 52.8, 39.7, 26.7, 19.4,−4.8, −5.0; HRMS (FAB+) calcd for C₂₀H₃₀O₃N₃ClSiI (M+H⁺): 550.0790.found: 550.0773.

3′-O-Allyl-7-deaza-7-iodo-2′-deoxyadenosine (16)

To a stirred solution of 15 (752 mg; 1.37 mmol) in anhydrous THF (32 mL)was added 1 M TBAF in THF solution (1.50 mL; 1.50 mmol) and the reactionwas stirred at room temperature for 1 h. The solvent was evaporated andthe residue was dissolved in 7 M methanolic ammonia (22 mL). Thesolution was stirred in an autoclave at 115-1.20° C. for 17 h. Afterevaporation of the solvent, the residue was purified by flash columnchromatography over silica gel using CH₃OH—CH₂Cl₂ (1:20) as the eluentto afford 16 as white solid (479 mg; 84% yield): ¹H NMR (400 MHz, CD₃OD)δ 8.08 (s, 1H, 2-H), 7.56 (s, 1H, 6-H), 6.45 (dd, J=5.8, 8.6 Hz, 1H,1′-H), 5.90-6.00 (m, 1H, CH₂CH═CH₂), 5.29-5.35 (dm, J=17.2 Hz, 1H, oneof CH₂CH═CH₂), 5.16-5.21 (dm, J=10.5 Hz, 1H, one of CH₂CH═CH₂), 4.28 (m,1H, 3′-H), 4.12 (m, 1H, 4′-H), 4.02-4.12 (m, 2H, CH₂CH═CH₂), 3.78 (dd,J=3.7, 12.1 Hz, 1H, one of 5′-H), 3.70 (dd, J=3.6, 12.1 Hz, 1H, one of5′-H), 2.53-2.61 (ddd, J=5.8, 8.6, 13.6 Hz, 1H, one of 2′-H), 2.41-2.47(ddd, J=2.0, 5.8, 13.5 Hz, 1H, one of 2′-H); ¹³C NMR (100 MHz, CD₃OD) δ158.5, 152.3, 150.3, 135.7, 128.8, 117.0, 105.3, 86.8, 86.4, 80.7, 71.0,63.7, 51.3, 38.8; HRMS (FAB+) calcd for C₁₄H₁₈O₃N₄I (M+H⁺): 417.0424.found: 417.0438.

3′-O-Allyl-7-deaza-7-[3-[(trifluoroacetyl)amino]-prop-1-ynyl]-2′-deoxyadenosine(17)

The procedure is the same as that of 7 and the crude product waspurified by flash column chromatography over silica gel using ethylacetate-hexane (1:1˜0) as the eluent to afford 17 as yellow solid (455mg; 90% yield): ¹H NMR (400 MHz, CD₃OD) δ 8.08 (s, 1H, 2-H), 7.60 (s,1H, 6-H), 6.41 (dd, J=5.8, 8.6 Hz, 1H, 1′-H), 5.89-6.00 (m, 1H,CH₂CH═CH₂), 5.29-5.35 (dm, J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.16-5.21(dm, J=10.4 Hz, 1H, one of CH₂CH═CH₂), 4.31 (s, 2H, C≡CCH₂), 4.29 (m,1H, 3′-H), 4.13 (m, 1H, 4′-H), 4.01-4.11 (m, 2H, CH₂CH═CH₂), 3.79 (dd,J=3.6, 12.1 Hz, 1H, one of 5′-H), 3.71 (dd, J=3.5, 12.1 Hz, 1H, one of5′-H), 2.54-2.62 (ddd, J=5.8, 8.6, 13.6 Hz, 1H, one of 2′-H), 2.42-2.48(ddd, J=1.9, 5.8, 13.6 Hz, 1H, one of 2′-H); ¹³C NMR (100 MHz, CD₃OD) δ158.8, 158.6 (q, J=38 Hz, COCF₃), 152.9, 149.6, 135.6, 128.1, 117.1 (q,J=284 Hz, COCF₃), 117.0, 104.5, 96.3, 87.3, 86.9, 86.8, 80.7, 77.0,71.0, 63.8, 38.7, 31.1; HRMS (FAB+) calcd for C₁₉H₂₁O₄N₅F₃ (M+H⁺):440.1546. found: 440.1544.

3′-O-Allyl-7-deaza-7-(3-aminoprop-1-ynyl)-2′-deoxyadenosine-5′-triphosphate(18)

The procedure is the same as that of preparing 9 to yield 17 ascolorless syrup: 1H NMR (300 MHz, D₂O) δ 8.02 (s, 1H, 2-H), 7.89 (s, 1H,6-H), 6.54 (t, J=6.6 Hz, 1H, 1′-H), 5.89-6.02 (m, 1H, CH₂CH═CH₂),5.30-5.39 (dm, J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.20-5.27 (dm, J=10.4Hz, 1H, one of CH₂CH═CH₂), 4.48 (s, 2H, C═CCH₂), 4.35 (m, 1H, 3′-H),4.05-4.17 (m, 4H, CH₂CH═CH₂ and 5′-H), 3.99 (m, 1H, 4′-H), 2.50-2.59 (m,2H, 2′-H); ³¹P NMR (121.4 MHz, D₂O) δ −6.1 (d, J=21.1 Hz, 1P, γ-P),−10.8 (d, J=18.8 Hz, 1P, α−P), −21.9 (t, J=19.9 Hz, 1P, β-P).

3′-O-allyl-dATP-ROX (19)

The coupling reaction of 18 with PC-ROX-NHS ester (Ref. 2b) afforded 19,following a similar procedure as the preparation of 10.3′-O-allyl-dATP-PC-ROX 19 was characterized by the primer extensionreaction and MALDI-TOF MS.

3) Synthesis of 3′-O-Allyl-dCTP-PC-Bodipy-650 as shown in FIG. 95′-O-(tert-Butyldimethylsilyl)-5-iodo-2′-deoxycytidine (21)

The procedure is the same as that of 5 and the crude product waspurified by flash column chromatography over silica gel usingCH₃OH—CH₂Cl₂ (1:20) as the eluent to afford 21 as white solid (1.18 g;89% yield): ¹H NMR (400 MHz, CD₃OD) δ 8.18 (s, 1H, 6-H), 6.17 (dd,J=5.8, 7.5 Hz, 1H, 1′-H), 4.34 (m, 1H, 3′-H), 4.04 (m, 1H, 4′-H), 3.93(dd, J=2.5, 11.6 Hz, 1H, one of 5′-H), 3.84 (dd, J=2.9, 11.6 Hz, 1H, oneof 5′-H), 2.41-2.48 (ddd, J=2.5, 5.8, 13.5 Hz, 1H, one of 2′-H),2.01-2.08 (ddd, J=5.9, 7.6, 13.5 Hz, 1H, one of 2′-H), 0.95 (s, 9H,C(CH₃)₃), 0.17 (s, 3H, one of SiCH₃), 0.16 (s, 3H, one of SiCH₃); ¹³CNMR (100 MHz, CD₃OD) δ 165.5, 156.8, 147.8, 89.4, 88.3, 72.8, 64.6,57.1, 43.1, 26.7, 19.4, −4.8, −4.9; HRMS (FAB+) calcd for C₁₅H₂₇O₄N₃SiI(M+H⁺): 468.0816. found: 468.0835.

3′-O-Allyl-5′-(tert-butyldimethylsilyl)-5-iodo-2′-deoxycytidine (22)

To a stirred solution of 21 (1.18 g; 2.52 mmol) in anhydrous THF (43 mL)was added 95% NaH powder (128 mg; 5.07 mmol). The suspension was stirredat room temperature for 45 min. Allyl bromide (240 μL, 2.79 mmol) wasthen added at 0° C. and the reaction was stirred at room temperature for14 h with exclusion of moisture. Saturated aqueous NaHCO₃ (10 mL) wasadded at 0° C. and stirred for 10 min. Most THF was evaporated and theresidue was dissolved in ethyl acetate (150 mL). The solution was washedby saturated aqueous NaHCO₃ and NaCl respectively, and dried overanhydrous Na₂SO₄. After evaporation of the solvent, the residue waspurified by flash column chromatography over silica gel using ethylacetate as the eluent to afford 22 as white solid (537 mg; 42% yield):¹H NMR (400 MHz, CD₃OD) δ 8.15 (s, 1H, 6-H), 6.12 (dd, J=5.6, 8.0 Hz,1H, 1′-H), 4.17 (m, 1H, 4′-H), 4.14 (m, 1H, 3′-H), 3.98-4.10 (m, 2H,CH₂CH═CH₂), 3.93 (dd, J=2.8, 11.5 Hz, 1H, one of 5′-H), 3.83 (dd, J=2.8,11.5 Hz, 1H, one of 5′-H), 2.53-2.60 (ddd, J=1.7, 5.6, 13.6 Hz, 1H, oneof 2′-H), 1.94-2.02 (ddd, J=5.9, 8.0, 13.6 Hz, 1H, one of 2′-H), 0.94(s, 9H, C(CH₃)₃), 0.17 (s, 3H, one of SiCH₃), 0.16 (s, 3H, one ofSiCH₃); ¹³C NMR (100 MHz, CD₃OD) δ 165.4, 156.7, 147.7, 135.5, 117.2,88.2, 87.0, 80.4, 70.9, 64.8, 57.3, 40.1, 26.7, 19.4, −4.7, −4.9; HRMS(FAB+) calcd for C₁₈H₃₁O₄N₃SiI (M+H⁺): 508.1129. found: 508.1123.

3′-O-allyl-5-iodo-2′-deoxycytidine (23)

To a stirred solution of 22 (537 mg; 1.06 mmol) in anhydrous THF (25 mL)was added 1 M TBAF in THF solution (1.17 mL; 1.17 20 mmol) and thereaction was stirred at room temperature for 1 h. The solvent wasevaporated and the residue was dissolved in ethyl acetate (100 mL). Thesolution was washed by saturated aqueous NaCl and dried over anhydrousNa₂SO₄. After evaporation of the solvent, the residue was purified byflash column chromatography over silica gel using CH₃OH—CH₂Cl₂ (1:10) asthe eluent to afford 23 as white crystals (287 mg; 69% yield): ¹H NMR(400 MHz, CD₃OD) δ 8.47 (s, 1H, 6-H), 6.15 (dd, J=6.2, 6.7 Hz, 1H,1′-H), 5.87-5.98 (m, 1H, CH₂CH═CH₂), 5.26-5.33 (dm, J=17.2 Hz, 1H, oneof CH₂CH═CH₂), 5.14-5.19 (dm, J=10.5 Hz, 1H, one of CH₂CH═CH₂), 4.18 (m,1H, 3′-H), 4.08 (m, 1H, 4′-H), 3.98-4.10 (m, 2H, CH₂CH═CH₂), 3.82 (dd,J=3.2, 13.0 Hz, 1H, one of 5′-H), 3.72 (dd, J=3.3, 13.0 Hz, 1H, one of5′-H), 2.44-2.51 (ddd, J=3.2, 6.0, 13.6 Hz, 1H, one of 2′-H), 2.07-2.15(m, 1H, one of 2′-H); ¹³C NMR (100 MHz, CD₃OD) δ 165.4, 156.9, 148.8,135.6, 117.0, 87.9, 86.9, 79.6, 71.2, 62.7, 57.2, 39.7; HRMS (FAB+)calcd for C₁₂H₁₇O₄N₃I (M+H⁺): 394.0264. found: 394.0274.

3′-O-Allyl-5-[3-[(trifluoroacetyl)amino]-prop-1-ynyl]-2′-deoxycytidine(24)

The procedure is the same as that of 7 and the crude product waspurified by flash column chromatography over silica gel usingCH₃OH—CH₂Cl₂ (0˜1:10) as the eluent to afford 24 as yellow crystals (252mg; 83% yield): ¹H NMR (400 MHz, CD₃OD) δ 8.31 (s, 1H, 6-H), 6.17 (dd,J=6.0, 7.3 Hz, 1H, 1′-H), 5.87-5.97 (m, 1H, CH₂CH═CH₂), 5.26-5.33 (dm,J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.15-5.19 (dm, J=10.4 Hz, 1H, one ofCH₂CH═CH₂), 4.31 (s, 2H, C≡CCH₂), 4.17 (m, 1H, 3′-H), 4.09 (m, 1H,4′-H), 3.98-4.10 (m, 2H, CH₂CH═CH₂), 3.80 (dd, J=3.4, 12.0 Hz, 1H, oneof 5′-H), 3.72 (dd, J=3.6, 12.0 Hz, 1H, one of 5′-H), 2.46-2.53 (ddd,J=2.9, 5.3, 13.6 Hz, 1H, one of 2′-H), 2.04-2.12 (m, 1H, one of 2′-H);¹³C NMR (100 MHz, CD₃OD) δ 166.0, 158.4 (q, J=38 Hz, COCF₃), 156.3,145.8, 135.6, 117.1 (q, J=284 Hz, COCF₃), 117.0, 91.9, 90.7, 88.0, 87.0,79.8, 75.5, 71.2, 62.8, 39.6, 31.0; HRMS (FAB+) calcd for C₁₇H₂₀O₅N₄F₃(M+H⁺): 417.1386. found: 417.1377.

3′-O-Allyl-5-(3-aminoprop-1-ynyl)-2′-deoxycytidine-5′-triphosphate (25)

The procedure is the same as that of preparing 9 to yield 25 ascolorless syrup: 1H NMR (300 MHz, D₂O) δ 8.43 (s, 1H, 6-H), 6.21 (t,J=6.7 Hz, 1H, 1′-H), 5.85-6.00 (m, 1H, CH₂CH═CH₂), 5.28-5.38 (dm, J=17.3Hz, 1H, one of CH₂CH═CH₂), 5.19-5.27 (dm, J=10.4 Hz, 1H, one ofCH₂CH═CH₂), 4.22-4.41 (m, 3H, 3′-H and C≡CCH₂), 4.05-4.18 (m, 3H, 4′-Hand CH₂CH═CH₂), 3.94-4.01 (m, 2H, 5′-H), 2.47-2.59 (m, 1H, one of 2′-H),2.20-2.32 (m, 1H, one of 2′-H); ³¹P NMR (121.4 MHz, D₂O) δ −7.1 (d,J=19.8 Hz, 1P, γ-P), −11.1 (d, J=19.1 Hz, 1P, α-P), −21.9 (t, J=19.5 Hz,IP, β-P).

3′ O-allyl-dCTP-PC-Bodipy-650 (26)

The coupling reaction of 25 with PC-Bodipy-650-NHS ester (Ref. 34b)afforded 26, following a similar procedure as the preparation of 10.3′-O-allyl-dCTP-PC-Bodipy-650 26 was characterized by the primerextension reaction and MALDI-TOF MS.

4) Synthesis of 3′-O-allyl-dUTP-PC-R6G as shown in FIG. 105′-O-(tert-butyldimethylsilyl)-5-iodo-2′-deoxyuridine (28)

The procedure is the same as that of 5 and the crude product waspurified by flash column chromatography over silica gel usingCH₃OH—CH₂Cl₂ (1:20) as the eluent to afford 28 as white solid (1.18 g;89% yield): ¹H NMR (400 MHz, CD₃OD) δ 8.17 (s, 1H, 6-H), 6.21 (dd,J=5.9, 7.9 Hz, 1H, 1′-H), 4.36 (m, 1H, 3′-H), 4.02 (m, 1H, 4′-H), 3.93(dd, J=2.4, 11.5 Hz, 1H, one of 5′-H), 3.85 (dd, J=2.9, 11.5 Hz, 1H, oneof 5′-H), 2.30-2.37 (ddd, J=2.3, 5.8, 13.4 Hz, 1H, one of 2′-H),2.08-2.15 (ddd, J=5.9, 7.9, 13.4 Hz, 1H, one of 2′-H), 0.96 (s, 9H,C(CH₃)₃), 0.19 (s, 3H, one of SiCH₃), 0.17 (s, 3H, one of SiCH₃). ¹³CNMR (100 MHz, CD₃OD) δ 162.4, 151.5, 145.8, 89.3, 87.2, 72.8, 68.7,64.6, 42.3, 26.8, 19.5, −4.8, −4.9. HRMS (FAB+) Calcd for C₁₅H₂₆O₅N₂SiI(M+H⁺): 469.0656. found: 469.0672.

3′-O-allyl-5′-O-(tert-butyldimethylsilyl)-5-iodo-2′-deoxyuridine (29)

The procedure is the same as that of 22 and the crude product waspurified by flash column chromatography over silica gel using ethylacetate-hexane (1:2.5) as the eluent to afford 29 as white solid (1.03g; 80% yield). ¹H NMR (400 MHz, CD₃OD) δ 8.15 (s, 1H, 6-H), 6.15 (dd,J=5.6, 8.3 Hz, 1H, 1′-H), 5.87-5.97 (m, 1H, CH₂CH═CH₂), 5.27-5.33 (dm,J=17.3 Hz, 1H, one of CH₂CH═CH₂), 5.16-5.21 (dm, J=10.4 Hz, 1H, one ofCH₂CH═CH₂), 4.1.3-4.18 (m, 2H, 3′-H and 4′-H), 3.99-4.10 (m, 2H,CH₂CH═CH₂), 3.92 (dd, J=2.7, 11.5 Hz, 1H, one of 5′-H), 3.84 (dd, J=2.7,11.5 Hz, 1H, one of 5′-H), 2.43-2.49 (ddd, J=1.7, 5.6, 13.6 15 Hz, 1H,one of 2′-H), 2.02-2.10 (ddd, J=5.6, 8.4, 13.6 Hz, 1H, one of 2′-H),0.96 (s, 9H, C(CH₃)₃), 0.18 (s, 3H, one of SiCH₃), 0.17 (s, 3H, one ofSiCH₃). ¹³C NMR (100 MHz, CD₃OD) δ 162.3, 151.4, 145.5, 135.5, 117.2,87.0, 86.8, 80.3, 70.9, 69.0, 64.8, 39.4, 26.8, 19.4, −4.7, −4.8. HRMS(FAB+) Calcd for C₁₈H₃₀O₅N₂SiI (M+H⁺): 509.0969. found: 509.0970.

3′-O-allyl-5′-(tert-butyldimethylsilyl)-5-[3-[(trifluoroacetyl)amino]-prop-1-ynyl]-2′-deoxyuridine(30)

The procedure is the same as that of 7 and the crude product waspurified by flash column chromatography over silica gel usingCH₃OH—CH₂Cl₂ (0-1:40) as the eluent to afford 30 as yellow crystals (786mg; 73% yield). ¹H NMR (400 MHz, CD₃OD) δ 8.11 (s, 1H, 6-H), 6.18 (dd,J=5.8, 7.9 Hz, 1H, 1′-H), 5.87-5.97 (m, 1H, CH₂CH═CH₂), 5.27-5.33 (dm,J=17.2 Hz, 1H, one of CH₂CH═CH₂), 5.16-5.21 (dm, J=10.4 Hz, 1H, one ofCH₂CH═CH₂), 4.27-4.32 (dd, J=17.7 Hz, 1H, one of C≡CCH₂), 4.21-4.27 (dd,J=17.7 Hz, 1H, one of C≡CCH₂), 4.14-4.18 (in, 2H, 3′-H and 4′-H),3.98-4.10 (m, 2H, CH₂CH═CH₂), 3.93 (dd, J=2.4, 11.5 Hz, 1H, one of5′-H), 3.84 (dd, J=2.2, 11.5 Hz, 1H, one of 5′-H), 2.44-2.50 (ddd,J=1.8, 5.7, 13.5 Hz, 1H, one of 2′-H), 2.04-2.12 (ddd, J=5.6, 8.0, 13.5Hz, 1H, one of 2′-H), 0.94 (s, 9H, C(CH) 3), 0.16 (s, 3H, one of SiCH₃),0.15 (s, 3H, one of SiCH₃). ¹³C NMR (100 MHz, CD₃OD) δ 164.1, 158.0 (q,J=37 Hz, COCF₃), 150.6, 144.3, 135.5, 117.3, 117.1 (q, J=284 Hz, COCF₃),99.5, 88.9, 87.2, 86.9, 80.3, 76.0, 71.0, 64.7, 39.6, 30.7, 26.6, 19.3,−5.0, −5.2. HRMS (FAB+) m/z: anal. Calcd for C₂₃H₃₃O₆N₃FSi (M+H⁺):532.2091. found: 532.2074.

3′-O-allyl-5-[3-[(trifluoroacetyl)amino]-prop-1-ynyl]-2′-deoxyuridine(31)

The procedure is the same as that of 23 and the crude product waspurified by flash column chromatography over silica gel using ethylacetate-hexane (3:1) as the eluent to afford 31 as yellow solid (302 mg;49% yield). ¹H NMR (400 MHz, CD₃OD) δ 8.29 (s, 1H, 6-H), 6.19 (dd,J=6.1, 7.4 Hz, 1H, 1′-H), 5.87-5.99 (m, 1H, CH₂CH═CH₂), 5.27-5.33 (dm,J=17.2 Hz, 1H, one of CH₂CH═CH₂), 5.15-5.20 (dm, J=10.4 Hz, 1H, one ofCH₂CH═CH₂), 4.27 (s, 2H, C≡CCH₂), 4.20 (m, 1H, 3′-H), 3.99-4.09 (m, 3H,4′-H and CH₂CH═CH₂), 3.80 (dd, J=3.3, 12.0 Hz, 1H, one of 5′-H), 3.72(dd, J=3.4, 12.0 Hz, 1H, one of 5′-H), 2.39-2.46 (ddd, J=2.6, 5.9, 13.7Hz, 1H, one of 2′-H), 2.14-2.22 (ddd, J=6.3, 7.5, 13.7 Hz, 1H, one of2′-H). ¹³C NMR (100 MHz, CD₃OD) δ 164.2, 158.0 (q, J=38 Hz, COCF₃),150.8, 145.3, 135.6, 117.2 (q, J=285 Hz, COCF₃), 117.1, 99.5, 88.3,87.1, 87.0, 79.9, 75.9, 71.2, 62.9, 39.0, 30.8. HRMS (FAB+) Calcd forC₁₇H₁₉O₆N₃F₃ (M+H⁺): 418.1226. found: 418.1213.

3′-O-allyl-5-(3-aminoprop-1-ynyl)-2′-deoxyuridine-5′-triphosphate (32)

The procedure is the same as that of preparing 9 to yield 32 ascolorless syrup: ¹H NMR (300 MHz, D₂O) δ 8.31 (s, 1H), 6.17 (t, 1H),5.81-5.90 (m, 1H), 5.18 (d, 1H), 5.14 (d, 1H), 4.34 (m, 2H), 4.03-4.15(m, 2H), 4.00 (d, 2H), 3.93 (s, 2H), 2.44-2.47 (m, 1H), 2.22-2.24 (m,1H). ³¹P NMR (121.4 MHz, D₂O) δ −5.90 (d, J=19.0 Hz, IP, γ-P), −11.43(d, J=20.0 Hz, IP, α-P), −22.25 (t, J=19.8 Hz, 1P, β-P).

3′-O-allyl-dUTP-PC-R6G (33)

The coupling reaction of 32 with PC-R6G-NHS ester (Ref. 34b) afforded33, following a similar procedure as the preparation of 10.3′-O-allyl-dCTP-PC-Bodipy-650 33 was characterized by the primerextension reaction and MALDI-TOF MS.

II. 3′-O-Allyl Modified Photocleavable Fluorescent Nucleotides asReversible Terminators for Primer Extension Reactions 1) PrimerExtension by Using 3′-O-Allyl-dGTP-PC-Bodipy-FL-510 (10) andPhotocleavage of the Extension Product 34

The polymerase extension reaction mixture consisted of 60 pmol of primer(5′-GTTGATGTACACATTGTCAA-3′), 80 pmol of 100-mer template(5′-TACCCGGAGGCCAAGTACGGCGGGTACGTCC-TTGACAATGTGTACATCAACATCACCTACCACCATGTCAGTCTCGGTTGGATCCTCTATTGTGTCCGGG-3′), 120 pmol of 3′-O-allyl-dGTP-PC-Bodipy-FL-510, 1×Thermopol reaction buffer (20 mM Tris-HCl/10 mM (NH₄)₂SO₄/10 mM KCl/2mM-MgSO₄/0.1% Triton X-100, pH 8.8, New England Biolabs), and 6 units of90N Polymerase (exo-)A485L/Y409V in a total volume of 20 μl. Thereaction consisted of 20 cycles at 94° C. for 20 sec, 46° C. for 40 sec,and 60° C. for 90 sec. After the reaction, a small portion of the DNAextension product was desalted by using ZipTip and analyzed by MALDI-TOFMS, which shows a dominant peak at m/z 7,052 corresponding to the DNAproduct 34. The rest of the product mixture was freeze-dried,resuspended in 200 μl of deionized water, and irradiated at 355 nm for10 sec to cleave the fluorophore from the DNA to yield product 35 andthen analyzed by MALDI-TOF MS.

Deallyation of Photocleaved DNA Extension Product 35

DNA product 35 (20 pmol) was added to a mixture of degassed 1× Thermopolreaction buffer (20 mM Tris-HCl/10 mM (NH₄)₂SO₄/10 mM KCl/2 mMMgSO₄/0.1% Triton X-100, pH 8.8, 1 μl), Na₂PdCl₄ in degassed H₂O (7 μl,23 20 nmol) and P(PhSO₃Na)₃ in degassed H₂O (10 μl, 176 nmol) to performdeallylation. The reaction mixture was then placed in a heating blockand incubated at 70° C. for 30 seconds to yield quantitativelydeallylated DNA product 36 and analyzed by MALDI-TOF MS.

Primer Extension Reaction Performed with the Deallylated DNA Product

The deallylated DNA product 36 was used as a primer in a single-baseextension reaction. The 20 μl reaction mixture consisted of 60 pmol ofthe deallylated product 36, 80 pmol of the 100-mer template(5′-TACCCGGAGGCCAAGTACGGCGGGTACGTCC-TTGACAATGTGTACATCAACATCACCTACCACCATGTCAGTCTCGGTTGGATCCTCTATTGTGTCCGGG-3′), 120 pmol of 3′-O-allyl-dGTP-PC-Bodipy-FL-510 (10), 6units of 9°N Polymerase (exo-)A485L/Y409V in a total volume of 20 μl.The reaction consisted of 20 cycles at 94° C. for 20 sec, 46° C. for 40sec, and 60° C. for 90 sec. The DNA extension product 37 was desalted byusing the ZipTip protocol, and a small portion was analyzed by usingMALDI-TOF MS. The remaining product was then irradiated with near-UVlight (355 nm) for 10 sec to cleave the fluorophore from the extendedDNA product. The resulting photocleavage product 38 was analyzed byusing MALDI-TOF MS. Finally, deallylation of the photocleavage product38 was performed using a Pd-catalyzed deallylation reaction resulting ina deallylated DNA product 39, which was then analyzed by MALDI-TOF MS.

2) Primer Extension with 3′-O-Allyl-dATP-PC-ROX (19), followed byPhotocleavage and Deallylation of the Extension Product

The polymerase extension reaction mixture consisted of 60 pmol of primer(5′-TAGATGACCCTGCCTTGTCG-3′), 80 pmol of 100-mer template(5′-GAAGGAGACACGCGGCCAGAGAGGGT-CCTGTCCGTGTTTGTGCGTGGAGTTCGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCTTTTCTCTTCGTTCTCCGT-3′), 120 pmol of 3′-O-allyl-dUTP-PC-R6G,120 pmol of 3′-O-allyl-dATP-PC-ROX, 120 pmol of3′-O-allyl-dGTP-PC-Bodipy-FL-510, 120 pmol of3′-O-allyl-dCTP-PC-Bodipy-650, 1× Thermopol reaction buffer (20 mMTris-HCl/10 mM (NH₄)₂SO₄/10 mM KCl/2 mM MgSO₄/0.1% Triton X-100, pH 8.8,New England Biolabs), and 6 units of 9°N Polymerase (exo-)A485L/Y409V ina total volume of 20 μl. The reaction consisted of 20 cycles at 94° C.for 20 sec, 55° C. for 40 sec, and 68° C. for 90 sec, which yielded DNAextension product 40. DNA extension product mixture was freeze-dried,resuspended in 200 μl of deionized water, and irradiated at 355 nm for10 sec to cleave the fluorophore from the DNA to yield DNA product 41and then analyzed by MALDI-TOF MS. Finally, deallylation of thephotocleavage product was performed using a Pd-catalyzed deallylationreaction resulting in a deallylated DNA product 42, which was thenanalyzed by MALDI-TOF MS.

3) Primer Extension with 3′-O-Allyl-dCTP-PC-Bodipy-650 (26), followed byPhotocleavage and Deallylation of the Extension Product

The polymerase extension reaction mixture consisted of 60 pmol of primer(5′-ACACAATAGAGGATCCAACCG AGA-3′), 80 pmol of 100-mer template(5′-TACCCGGAGGCCAAGTACGGCGGGTACGTCCTTGACAATGTGTACATCAACATCACCTACCACCATGTCAGTCTCGGTTGGATCCTCTATTGTGTCCGGG-3′), 120 pmol of 3′-O-allyl-dUTP-PC-R6G, 120 pmolof 3′-O-allyl-dATP-PC-ROX, 120 pmol of 3′-O-allyl-dGTP-PC-Bodipy-FL-510,120 pmol of 3′-O-allyl-dCTP-PC-Bodipy-650, 1× Thermopol reaction buffer(20 mM Tris-HCl/10 mM (NH₄)₂SO₄/10 mM KCl/2 mM MgSO₄/0.1% Triton X-100,pH 8.8, New England Biolabs), and 6 units of 9°N Polymerase(exo-)A485L/Y409V in a total volume of 20 μl. The reaction consisted of20 cycles at 94° C. for 20 sec, 64° C. for 40 sec, and 72° C. for 90sec, which yielded DNA extension product 43. DNA extension productmixture was freeze-dried, resuspended in 200 μl of deionized water, andirradiated at 355 nm for 10 sec to cleave the fluorophore from the DNAto yield DNA product 44 and then analyzed by MALDI-TOF MS. Finally,deallylation of the photocleavage product was performed using aPd-catalyzed deallylation reaction resulting in a deallylated DNAproduct 45, which was then analyzed by MALDI-TOF MS.

4) Primer Extension with 3′-O-Allyl-dUTP-PC-R6G (33), followed byPhotocleavage and Deallylation of the Extension Product

The polymerase extension reaction mixture consisted of 60 pmol of primer(5′-GATAGGACTCATCACCA-3′), 80 pmol of 100-mer template(5′-GAAGGAGACACGCGGCCAGAGAGGGTCCTGTCCGTGTTTGT GCGTGGAGTTCGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCTTT TCTCTTCGTTCTCCGT-3′),120 pmol of 3′-O-allyl-dUTP-PC-R6G, 120 pmol of 3′-O-allyl-dATP-PC-ROX,120 pmol of 3′-O-allyl-dGTP-PC-Bodipy-FL-510, 120 pmol of3′-O-allyl-dCTP-PC-Bodipy-650, 1× Thermopol reaction buffer (20 mMTris-HCl/10 mM (NH₄)₂SO₄/10 mM KCl/2 mM MgSO₄/0.1% Triton X-100, pH 8.8,New England Biolabs), and 6 units of 9°N Polymerase (exo-)A485L/Y409V ina total volume of 20 μl. The reaction consisted of 20 cycles at 94° C.for sec, 46° C. for 40 sec, and 60° C. for 90 sec, which yielded DNAextension product 46. DNA extension product mixture was freeze-dried,resuspended in 200 μl of deionized water, and irradiated at 355 nm for10 sec to cleave the fluorophore from the DNA to yield DNA product 47and then analyzed by MALDI-TOF MS. Finally, deallylation of thephotocleavage product was performed using a Pd-catalyzed deallylationreaction resulting in a deallylated DNA product 48, which was thenanalyzed by MALDI-TOF MS.

Example 3 Four-Color DNA Sequencing by Synthesis on a Chip UsingPhotocleavable Fluorescent Nucleotides Synopsis

In this example, 4-color DNA sequencing by synthesis (SBS) on a chipusing four photocleavable fluorescent nucleotide analogues(dGTP-PC-Bodipy-FL-510, dUTP-PC-R6G, dATP-PC-ROX, anddCTP-PC-Bodipy-650) is demonstrated. Each nucleotide analogue consistsof a different fluorophore attached to the 5-position of the pyrimidines(C and U) and the 7-position of the purines (G and A) through aphotocleavable 2-nitrobenzyl linker. After verifying that thesenucleotides could be successfully incorporated into a growing DNA strandin a solution-phase polymerase reaction and the fluorophore could becleaved using laser irradiation (X-355 nm) in 10 seconds, an SBSreaction was then performed on a chip which contains a self-priming DNAtemplate covalently immobilized using 1,3-dipolar azide-alkynecycloaddition. The DNA template was produced by a polymerase chainreaction using an azido-labeled primer and the self-priming moiety wasattached to the immobilized DNA template by enzymatic ligation. Eachcycle of SBS consists of the incorporation of the photocleavablefluorescent nucleotide into the DNA, detection of the fluorescent signaland photocleavage of the fluorophore. The entire process was repeated toidentify 12 continuous bases in the DNA template. These resultsdemonstrate that photocleavable fluorescent nucleotide analogues can beincorporated accurately into a growing DNA strand during a polymerasereaction in solution phase as well as on a chip. Moreover, all 4fluorophores can be detected and then efficiently cleaved using near-UVirradiation, thereby allowing continuous identification of the DNAtemplate sequence. Optimization of the steps involved increases thereadlength.

Results

DNA sequencing is a fundamental tool for biological science. Thecompletion of the Human Genome Project has set the stage for screeninggenetic mutations to identify disease genes on a genome-wide scale (42).Accurate high-throughput DNA sequencing methods are needed to explorethe complete human genome sequence for applications in clinical medicineand health care. Recent studies have indicated that an important routefor identifying functional elements in the human genome involvessequencing the genomes of many species representing a wide sampling ofthe evolutionary tree (43). To overcome the limitations of the currentelectrophoresis-based sequencing technology (44-47), a variety of newDNA-sequencing methods have been investigated. Such approaches includesequencing by hybridization (48), mass spectrometry based sequencing(49-51), sequence-specific detection of single-stranded DNA usingengineered nanopores (52). More recently, DNA sequencing by synthesis(SBS) approaches such as pyrosequencing (53), sequencing of single DNAmolecules (54) and polymerase colonies (55) have been widely explored.

The concept of DNA sequencing by synthesis was revealed in 1988 (56).This approach involves detection of the identity of each nucleotideimmediately after its incorporation into a growing strand of DNA in apolymerase reaction. Thus far, no complete success has been reported inusing such a system to sequence DNA unambiguously. An SBS approach wasproposed using photocleavable fluorescent nucleotide analogues on asurface in 2000 (57). In this approach, modified nucleotides are used asreversible terminators, in which a different fluorophore with a distinctfluorescent emission is linked to each of the 4 bases through aphotocleavable linker and the 3′-OH group is capped by a small chemicalmoiety. DNA polymerase incorporates only a single nucleotide analoguecomplementary to the base on a DNA template covalently linked to asurface. After incorporation, the unique fluorescence emission isdetected to identify the incorporated nucleotide and the fluorophore issubsequently removed photochemically. The 3′-OH group is then chemicallyregenerated, which allows the next cycle of the polymerase reaction toproceed. Since the large surface on a DNA chip can have a high densityof different DNA templates spotted, each cycle can identify many basesin parallel, allowing the simultaneous sequencing of a large number ofDNA molecules. The advantage of using photons as reagents for initiatingphotoreactions to cleave the fluorophore is that no additional chemicalreagents are required to be introduced into the system and cleanproducts can be generated with no need for subsequent purification. Ithas previously been established the feasibility of performing SBS on achip using a synthetic DNA template and photocleavable pyrimidinenucleotides (C and U) (58). As further development of this approach,here the design and synthesis of 4 photocleavable nucleotide analogues(A, C, G, U) is reported, each of which contains a unique fluorophorewith a distinct fluorescence emission. Initially, it is established thatthese nucleotides are good substrates for DNA polymerase in asolution-phase DNA extension reaction and that the fluorophore can beremoved with high speed and efficiency by laser irradiation (X-355 nm).Subsequently, SBS was performed using these 4 photocleavable nucleotideanalogues to identify the sequence of a DNA template immobilized on achip. The DNA template was produced by PCR using an azido-labeledprimer, and was immobilized on the surface of the chip with 1,3-dipolarazide-alkyne cycloaddition chemistry. A self-priming moiety was thencovalently attached to the DNA template by enzymatic ligation to allowthe polymerase reaction to proceed on the DNA immobilized on thesurface.

Materials and Methods

All chemicals were purchased from Sigma-Aldrich unless otherwiseindicated. 1H NMR spectra were recorded on a Bruker 400 spectrometer.High-resolution MS (HRMS) data were obtained by using a JEOL JMS HX 110Amass spectrometer. Mass measurement of DNA was made on a Voyager DEmatrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF)mass spectrometer (Applied Biosystems). Photolysis was performed using aSpectra Physics GCR-150-30 Nd-YAG laser that generates light pulses at355 nm (ca. 50 mJ/pulse, pulse length ca. 7 ns) at a frequency of 30 Hzwith a light intensity at ca. 1.5 W/cm2. The scanned fluorescenceemission images were obtained by using a ScanArray Express scanner(Perkin-Elmer Life Sciences) equipped with four lasers with excitationwavelengths of 488, 543, 594, and 633 nm and emission filters centeredat 522, 570, 614, and 670 nm.

Synthesis of Photocleavable Fluorescent Nucleotides

Photocleavable fluorescent nucleotides dGTP-PC-Bodipy-FL-510,dUTP-PC-R6G, dATP-PC-ROX and dCTP-PC-Bodipy-650 (FIG. 16) weresynthesized according to FIG. 17 using a similar method as reportedpreviously (57). A photocleavable linker (PC-Linker)1-[5-(aminomethyl)-2-nitrophenyl]ethanol was reacted with the NHS esterof the corresponding fluorescent dye to produce an intermediate PC-Dye,which was converted to a PC-Dye NHS ester by reacting with N,N′-disuccinimidyl carbonate. The coupling reaction between the differentPC-Dye NHS esters and the amino nucleotides (dATP-NH2 and dGTP-NH2 fromPerkin-Elmer; dUTP-NH2 from Sigma; dCTP-NH2 from TriLinkBioTechnologies) produced the 4 photocleavable fluorescent nucleotides.

DNA polymerase reaction using 4 photocleavable fluorescent nucleotideanalogues in solution. Four nucleotide analogues were characterized,dGTP-PC-Bodipy-FL-510, dUTP-PC-R6G, dATP-PC-ROX and dCTP-PC-Bodipy-650by performing four continuous DNA-extension reactions sequentially usinga primer (5′-AGAGGATCCAACCGAGAC-3′) and a synthetic DNA template(5′-GTGTACATCAACATCACCTACCACCATGTCAGTCTCGGTTGGAT-CCTCTATTGTGTCCGG-3′)corresponding to a portion of exon 7 of the human p53 gene (FIG. 18).The four nucleotides in the template immediately adjacent to theannealing site of the primer were 3′-ACTG-5′. First, a polymeraseextension reaction using dUTP-PC-R6G along with the primer and thetemplate was performed producing a single base extension product. Thereaction mixture for this, and all subsequent extension reactions,consisted of 80 pmol of template, 50 pmol of primer, 80 pmol of theparticular photocleavable fluorescent nucleotide, 1× Thermo Sequenasereaction buffer, and 4 U of Thermo Sequenase DNA polymerase (AmershamBiosciences) in a total volume of 20 L. The reaction consisted of 25cycles at 94° C. for 20 sec, 48° C. for 40 sec, and 60° C. for 75 sec.Subsequently, the extension product was purified by using reverse-phaseHPLC. An Xterra MS C18 (4.6×50-mm) column (Waters) was used for the HPLCpurification. Elution was performed over 120 minutes at a flow rate of0.5 mL/min with the temperature set at 50° C. by using a linear gradient(12-34.5%) of methanol in a buffer consisting of 8.6 mM triethylamineand 100 mM hexafluoroisopropyl alcohol (pH 8.1). The fraction containingthe desired DNA product was collected and freeze-dried for analysisusing MALDI-TOF mass spectrometry. For photocleavage, the purified DNAextension product bearing the fluorescent nucleotide analogue wasresuspended in 200 μL of deionized water. The mixture was irradiated for10 seconds in a quartz cell with path lengths of 1.0 cm employing aNd-YAG laser at 355 nm and then analyzed by MALDI-TOF MS. Afterphotocleavage, the DNA product with the fluorophore removed was used asa primer for a second extension reaction using dGTP-PC-Bodipy-FL-510.The second extended product was then purified by HPLC and photolyzed.The third extension using dATP-PC-ROX and the fourth extension usingdCTP-PC-Bodipy-650 were carried out in a similar manner using thepreviously extended and photocleaved product as the primer.

PCR Amplification to Produce Azido-Labeled DNA Template

An azido-labeled PCR product was obtained using a 100-bp template(5′-AGCGACTGCTATCATGTCATATCGACGTGCTCACTAGCTCTACATATGCGTGCGTGATCAGATGACGTATCGATGCTGACTATAGTCTCCCATGCGAGTG-3′), a 24-bp azido-labeledforward primer (5′-N3-AGCGACTGCTATCATGTCATATCG-3′), and a 24-bpunlabeled reverse primer (5′-CACTCGCATGGGAGACTATAGTCA-3′). In a totalreaction volume of 50 μL, 1 pmol of template and 30 pmol of forward andreverse primers were mixed with 1 U of AccuPrime Pfx DNA polymerase and5 μL of 10× AccuPrime Pfx reaction mix (Invitrogen) containing 1 mM ofMgSO₄ and 0.3 mM of dNTP. The PCR reaction consisted of an initialdenaturation step at 95° C. for 1 min, followed by 38 cycles at 94° C.for 15 sec, 63° C. for 30 sec, 68° C. for 30 sec. The product waspurified using a 96 QiAquick multiwell PCR purification kit (Qiagen) andthe quality was checked using 2% agarose gel electrophoresis in 1×TAEbuffer. The concentration of the purified PCR product was measured usinga Perkin-Elmer Lambda 40 UV-Vis spectrophotometer.

Construction of a Self-Priming DNA Template on a Chip by EnzymaticLigation

The amino-modified glass slide (Sigma) was functionalized to contain aterminal alkynyl group as described previously (57). The azido-labeledDNA product generated by PCR was dissolved in DMSO/H₂O (1/3, v/v) toobtain a 20 μM solution. 5 L of the DNA solution was mixed with CuI (10nmol, 100 eq.) and N,N-diisopropyl-ethylamine (DIPEA) (10 nmol, 100 eq.)and then spotted onto the alkynyl-modified glass surface in the form of6 L drops. The glass slide was incubated in a humid chamber at roomtemperature for 24 hr, washed with deionized water (dH₂O) and SPSCbuffer (50 mM sodium phosphate, 1 M NaCl, pH 6.5) for 1 hr (57), andfinally rinsed with dH₂O. To denature the double stranded PCR-amplifiedDNA to remove the non-azido-labeled strand, the glass slide was immersedinto 0.1 M NaOH solution for 10 min and then washed with 0.1 M NaOH anddH₂O, producing a single stranded DNA template that is immobilized onthe chip. For the enzymatic ligation of a self-priming moiety to theimmobilized DNA template on the chip, a 5′-phosphorylated 40-bp loopprimer (5′-PO3-GCTGAATTCCGCGTTCGCGGAATTCAGCCACTCGCATGGG-3′) wassynthesized. This primer contained a thermally stable loop sequence3′-G(CTTG)C-5′, a 12-bp stem, and a 12-bp overhanging end that would beannealed to the immobilized single stranded template at its 3′-end. A 10μL solution consisting of 100 pmol of the primer, 10 U of Taq DNAligase, 0.1 mM NAD, and 1× reaction buffer (New England Biolabs) wasspotted onto a location of the chip containing the immobilized DNA andincubated at 45° C. for 4 hr. The glass slide was washed with dH₂O, SPSCbuffer, and again with dH₂O. The formation of a stable hairpin wasascertained by covering the entire surface with 1× reaction buffer (26mM Tris-HCl/6.5 mM MgCl₂, pH 9.3), incubating it in a humid chamber at94° C. for 5 min to dissociate any partial hairpin structure, and thenslowly cooling down to room temperature for reannealing.

SBS Reaction on a Chip with Four Photocleavable Fluorescent NucleotideAnalogues.

One microliter of a solution consisting of dATP-PC-ROX (60 pmol), 2 U ofThermo Sequenase DNA polymerase, and 1× reaction buffer was spotted onthe surface of the chip, where the self-primed DNA moiety wasimmobilized. The nucleotide analogue was allowed to incorporate into theprimer at 72° C. for 5 min. After washing with a mixture of SPSC buffer,0.1% SDS, and 0.1% Tween 20 for 10 min, the surface was rinsed with dH₂Oand ethanol successively, and then scanned with a ScanArray Expressscanner to detect the fluorescence signal. To perform photocleavage, theglass chip was placed inside a chamber (50×50×50 mm) filled withacetonitrile/water (1/1, v/v) solution and irradiated for 1 min with theNd-YAG laser at 355 nm. The light intensity applied on the glass surfacewas ca. 1.5 W/cm². After washing the surface with dH₂O and ethanol, thesurface was scanned again to compare the intensity of fluorescence afterphotocleavage with the original fluorescence intensity. This process wasfollowed by the incorporation of dGTP-PC-Bodipy-FL-510, with thesubsequent washing, fluorescence detection, and photocleavage processesperformed as described above. The same cycle was repeated 10 more timesusing each of the four photocleavable fluorescent nucleotide analoguescomplementary to the base on the template. For a negative controlexperiment, 1 μL solution containing dATP-PC-ROX (60 pmol), and 1×reaction buffer was added on to the DNA immobilized on the chip in theabsence of DNA polymerase and then incubated at 72° C. for 5 min,followed by the same washing and detection steps as above.

RESULTS AND DISCUSSION

To demonstrate the feasibility of carrying out DNA sequencing bysynthesis on a chip, four photocleavable fluorescent nucleotideanalogues (dGTP-PC-Bodipy-FL-510, dUTP-PC-R6G, dATP-PC-ROX, anddCTP-PC-Bodipy-650) (FIG. 16) were synthesized according to FIG. 17using a similar procedure as reported previously (57). Modified DNApolymerases have been shown to be highly tolerant to nucleotidemodifications with bulky groups at the 5-position of pyrimidines (C andU) and the 7-position of purines (A and G) (59, 60). Thus, each uniquefluorophore was attached to the 5 position of C/U and the 7 position ofA/G through a photocleavable 2-nitrobenzyl linker.

In order to verify that these fluorescent nucleotides are incorporatedaccurately in a base-specific manner in a polymerase reaction, fourcontinuous steps of DNA extension and photocleavage by near UVirradiation were carried out in solution as shown in FIG. 18. Thisallows the isolation of the DNA product at each step for detailedmolecular structure characterization as shown in FIG. 19. The firstextension product 5′-U(PC-R6G)-3′ 1 was purified by HPLC and analyzedusing MALDI-TOF MS [FIG. 19(1)]. This product was then irradiated at 355nm using an Nd-YAG laser for 10 seconds and the photocleavage product 2was also analyzed using MALDI-TOF MS [FIG. 19(2)]. Near UV lightabsorption by the aromatic 2-nitrobenzyl linker causes reduction of the2-nitro group to a nitroso group and an oxygen insertion into thecarbon-hydrogen bond followed by cleavage and decarboxylation (61). Ascan be seen from FIG. 19(1), the MALDI-TOF MS spectrum consists of adistinct peak at m/z 6536 corresponding to the DNA extension product5′-U(PC-R6G)-3′ (1), which confirms that the nucleotide analogue can beincorporated base specifically by DNA polymerase into a growing DNAstrand. The small peak at m/z 5872 corresponding to the photocleavageproduct is due to the partial cleavage caused by the nitrogen laserpulse (337 nm) used in MALDI ionization. For photocleavage, a Nd-YAGlaser was used to irradiate the DNA product carrying the fluorescentnucleotide for 10 seconds at 355 nm to cleave the fluorophore from theDNA extension product. FIG. 19(2) shows the photocleavage result of theabove DNA product. The peak at m/z 6536 has completely disappeared whilethe peak corresponding to the photocleavage product 5′-U (2) appears asthe sole dominant peak at m/z 5872, which establishes that laserirradiation completely cleaves the fluorophore with high speed andefficiency without damaging the DNA. The next extension reaction wascarried out using this photocleaved DNA product as a primer along withdGTP-PC-Bodipy-FL-510 to yield an extension product5′-UG(PC-Bodipy-FL-510)-3′ (3). As described above, the extensionproduct 3 was purified, analyzed by MALDI-TOF MS producing a dominantpeak at m/z 6751 [FIG. 19(3)], and then photocleaved for further MSanalysis yielding a single peak at m/z 6255 (product 4) [FIG. 19(4)].The third extension using dATP-PC-ROX to yield 5′-UGA(PC-ROX)-3′ (5),the fourth extension using dCTP-PC-Bodipy-650 to yield5′-UGAC(PC-Bodipy-650)-3′ (7) and their photocleavage to yield products6 and 8 were similarly carried out and analyzed by MALDI-TOF MS as shownin FIGS. 19(5), 19(6), 19(7) and 19(8). These results demonstrate thatthe above-synthesized four photocleavable fluorescent nucleotideanalogues can successfully incorporate into the growing DNA strand in apolymerase reaction, and the fluorophore can be efficiently cleaved bynear UV irradiation, which makes it feasible to use them for SBS on achip.

The photocleavable fluorescent nucleotide analogues were then used in anSBS reaction to identify the sequence of the DNA template immobilized ona solid surface as shown in FIG. 20. A site-specific 1,3-dipolarcycloaddition coupling chemistry was used to covalently immobilize theazido-labeled double-stranded PCR products on the alkynyl-functionalizedsurface in the presence of a Cu(I) catalyst. Previously, it has shownhave shown that DNA is successfully immobilized on the glass surface bythis chemistry and evaluated the functionality of the surface-bound DNAand the stability of the array using a primer extension reaction (57).The surface-immobilized double stranded PCR product was denatured usinga 0.1 M NaOH solution to remove the complementary strand without theazido group, thereby generating a single-stranded PCR template on thesurface. Then, a 5′-phosphorylated self-priming moiety (loop primer) wasligated to the 3′-end of the above single stranded DNA template usingTaq DNA ligase (21). The structure of the loop primer was designed tobear a thermally stable loop (22) and stem sequence with a meltingtemperature of 89° C. The 12-bp overhanging portion of the loop primerwas made complementary to the 12-bp sequence of the template at its 3′end to allow the Taq DNA ligase to seal the nick between the5′-phosphate group of the loop primer and the 3′-hydroxyl group of thesingle-stranded DNA template. This produces a unique DNA moiety that canself-prime for the synthesis of a complementary strand. The ligation wasfound to be in quantitative yield in a parallel solution-phase reactionusing the same primer and single-stranded DNA template.

The principal advantage offered by the use of a self-priming moiety ascompared to using separate primers and templates is that the covalentlinkage of the primer to the template in the self-priming moietyprevents any possible dissociation of the primer from the template undervigorous washing conditions. Furthermore, the possibility of misprimingis considerably reduced and a universal loop primer can be used for allthe templates allowing enhanced accuracy and ease of operation. SBS wasperformed on the chip-immobilized DNA template using the 4photocleavable fluorescent nucleotide analogues and the results areshown in FIG. 21. The structure of the self-priming DNA moiety is shownschematically in the upper panel, with the first 12 nucleotide sequenceimmediately after the priming site. The sequencing reaction on the chipwas initiated by extending the self-priming DNA using dATP-PC-ROX(complementary to the T on the template), and Thermo Sequenase DNApolymerase. After washing, the extension of the primer by a singlefluorescent nucleotide was confirmed by observing an orange signal (theemission signal from ROX) in a microarray scanner [FIG. 21(1)]. Afterdetection of the fluorescent signal, the surface was irradiated at 355nm for 1 min using an Nd-YAG laser to cleave the fluorophore. Thesurface was then washed, and a negligible residual fluorescent signalwas detected to confirm complete photocleavage of the fluorophore [FIG.21(2)]. This was followed by incorporation of the next fluorescentnucleotide complementary to the subsequent base on the template. Theentire process of incorporation, detection and photocleavage wasperformed multiple times using the four photocleavable fluorescentnucleotide analogues to identify 12 successive bases in the DNAtemplate. The integrated fluorescence intensity on the spot, obtainedfrom the scanner software, indicated that the incorporation efficiencywas over 90% and more than 97% of the original fluorescence signal wasremoved by photocleavage. A negative control experiment consisting ofincubating the self-priming DNA moiety with dATP-PC-ROX in the absenceof DNA polymerase and washing the surface showed that negligiblefluorescence remained as compared to that of FIG. 21(1).

In summary, synthesis and characterization of four photocleavablefluorescent nucleotide analogues are disclosed here, and their use toproduce 4-color DNA sequencing data on a chip. These nucleotides havebeen shown to be excellent substrates for the DNA polymerase and thefluorophore could be cleaved efficiently using near UV irradiation. Thisis important with respect to enhancing the speed of each cycle in SBSfor high throughput DNA analysis. It has also been demonstrated that aPCR-amplified DNA template can be ligated with a self-priming moiety andits sequence can be accurately identified in a DNA polymerase reactionon a chip, indicating that a PCR product from any organism can bepotentially used as a template for the SBS system in the future. Themodification of the 3′-OH of the photocleavable fluorescent nucleotidewith a small chemical group to allow reversible termination is reportedin (58). The library of photocleavable fluorescent nucleotides reportedhere should also facilitate the development of single molecule DNAsequencing approaches. Thus, by further improving the readlength andincorporation efficiency, this approach potentially can be developedinto a high-throughput DNA-analysis system for biological research andmedical applications.

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1. A method for making a 3′-O-allyl modified nucleotide analoguecomprising performing the steps set forth in one of FIG. 7, FIG. 8, FIG.9, or FIG.
 10. 2-4. (canceled)
 5. A method for determining the sequenceof a DNA comprising performing the following steps for each residue ofthe DNA to be sequenced: (a) contacting the DNA with a DNA polymerase inthe presence of (i) a primer and (ii) four fluorescent nucleotideanalogues under conditions permitting the DNA polymerase to catalyze DNAsynthesis, wherein (1) the nucleotide analogues consist of an analogueof dGTP, an analogue of dCTP, an analogue of dTTP or dUTP, and ananalogue of dATP, (2) each nucleotide analogue comprises (i) a baseselected from the group consisting of adenine, guanine, cytosine,thymine, or uracil and analogues thereof, (ii) a deoxyribose, (iii) afluorophore photocleavably attached to the base, and (iv) an allylmoiety bound to the 3′-oxygen of the deoxyribose, so that a nucleotideanalogue complementary to the residue being sequenced is bound to theDNA by the DNA polymerase, and (3) each of the four analogues has apredetermined fluorescence wavelength which is different than thefluorescence wavelengths of the other three analogues; (b) removingunbound nucleotide analogues; (c) determining the identity of the boundnucleotide analogues; and (d) following step (c), except with respect tothe final DNA residue to be sequenced, (i) chemically cleaving from thebound nucleotide analogue the allyl moiety bound to the 3′-oxygen atomof the deoxyribose and (ii) photocleaving the fluorophore from the boundnucleotide analogue, wherein steps (d)(i) and (d) (ii) can be performedconcurrently or in any order, and step (d)(1) is performed using a Pdcatalyst at a pH of about 8.8, thereby determining the sequence of theDNA.
 6. The method of claim 5, wherein chemically cleaving the allylmoiety bound to the 3′-oxygen atom is performed using Na₂PdCl₄.
 7. Themethod of claim 5, wherein the primer is a self-priming moiety.
 8. Themethod of claim 5, wherein the DNA is bound to a solid substrate.
 9. Themethod of claim 8, wherein the DNA is bound to the solid substrate via1,3-dipolar azide-alkyne cycloaddition chemistry.
 10. The method ofclaim 8, wherein about 1000 or fewer copies of the DNA are bound to thesolid substrate.
 11. The method of claim 5, wherein the four fluorescentnucleotide analogues are 3′-O-allyl-dGTP-PC-Bodipy-FL-510,3′-O-allyl-dATP-PC-ROX, 3′-O-allyl-dCTP-PC-Bodipy-650 and3′-O-allyl-dUTP-PC-R6G.
 12. The method of claim 5, wherein the DNApolymerase is a 9°N polymerase.
 13. A method for removing an allylmoiety from the 3′-oxygen of a nucleotide analogue's deoxyribose moietycomprising the step of contacting the nucleotide analogue with a Pdcatalyst at a pH of about 8.8.
 14. The method of claim 13, wherein thePd catalyst is Na₂PdCl₄.